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Document Overview

This document outlines a common workflow for using the Open Specy package and highlights some topics that users are often requesting a tutorial on. If the document is followed sequentially from beginning to end, the user will have a better understanding of every procedure involved in using the Open Specy R package as a tool for interpreting spectra. It takes approximately 45 minutes to read through and follow along with this standard operating procedure the first time. Afterward, knowledgeable users should be able to thoroughly analyze spectra at an average speed of 1 min-1 or faster with the new batch and automated procedures.

The Open Specy R package is the backbone of the Shiny app. The choice is yours as to which you start with, we use both on a regular basis. The tutorial will talk through the R functions you can use to programatically analyze spectra. If you are looking for a tutorial about how to use the app see this tutorial.

Installation

After installing the R package, you just need to read in the library.

Running the App

To get started with the Open Specy user interface, access https://openanalysis.org/openspecy/ or start the Shiny GUI directly from your own computer in R. If you are looking for a tutorial about how to use the app see this tutorial.

Read Data

The following line of code will read in your data when using the package and interprets which reading function to use based on the file extension.

read_any("path/to/your/data")

These file type specific functions will also read in spectral data accordingly if you have a particular format in mind.

read_text(".csv")
read_asp(".asp")
read_opus(".0")

Open Specy allows for upload of native Open Specy .y(a)ml, .json, or .rds files. In addition, .csv, .asp, .jdx, .0, .spa, .spc, and .zip files can be imported. .zip files can either contain multiple files with individual spectra in them of the non-zip formats or it can contain a .hdr and .dat file that form an ENVI file for a spectral map. Open Specy and .csv files should always load correctly but the other file types are still in development, though most of the time these files work perfectly. If uploading a .csv file, label the column with the wavenumbers wavenumber and name the column with the intensities intensity. Wavenumber units must be cm-1. Any other columns are not used by the software. Always keep a copy of the original file before alteration to preserve metadata and raw data for your records.

It is best practice to cross check files in the proprietary software they came from and Open Specy before use in Open Specy. Due to the complexity of some proprietary file types, we haven’t been able to make them fully compatible yet. If your file is not working, please contact the administrator and share the file so that we can work on integrating it.

The specific steps to converting your instrument’s native files to .csv can be found in its software manual or you can check out Spectragryph, which supports many spectral file conversions. For instructions, see Spectragryph Tutorial.

If you don’t have your own data, you can use a test dataset.

data("raman_hdpe")

We also have many onboard files that you can call to test different formats:

spectral_map <- read_extdata("CA_tiny_map.zip") |> 
  read_any() # preserves some metadata
asp_example <- read_extdata("ftir_ldpe_soil.asp") |> 
  read_any() 
ps_example <- read_extdata("ftir_ps.0") |> 
  read_any() # preserves some metadata
csv_example <- read_extdata("raman_hdpe.csv") |> 
  read_any()
json_example <- read_extdata("raman_hdpe.json") |> 
  read_any() # read in exactly as an OpenSpecy object

You will notice now that the R package reads in files into an object with class OpenSpecy. This is a class we created for high throughput spectral analysis which now also preserves spectral metadata. You can even create these from scratch if you’d like.

scratch_OpenSpecy <- as_OpenSpecy(x = seq(1000,2000, by = 5), 
                                  spectra =  data.frame(runif(n = 201)), 
                                  metadata = list(file_name = "fake_noise")) 

Open Specy objects are lists with three components, wavenumber is a vector of the wavenumber values for the spectra and corresponds to the rows in spectra which is a data.table where each column is a set of spectral intensities. metadata is a data.table which holds additional information about the spectra. Each row corresponds to a column in spectra.

# Access the wavenumbers
scratch_OpenSpecy$wavenumber
#>   [1] 1000 1005 1010 1015 1020 1025 1030 1035 1040 1045 1050 1055 1060 1065 1070
#>  [16] 1075 1080 1085 1090 1095 1100 1105 1110 1115 1120 1125 1130 1135 1140 1145
#>  [31] 1150 1155 1160 1165 1170 1175 1180 1185 1190 1195 1200 1205 1210 1215 1220
#>  [46] 1225 1230 1235 1240 1245 1250 1255 1260 1265 1270 1275 1280 1285 1290 1295
#>  [61] 1300 1305 1310 1315 1320 1325 1330 1335 1340 1345 1350 1355 1360 1365 1370
#>  [76] 1375 1380 1385 1390 1395 1400 1405 1410 1415 1420 1425 1430 1435 1440 1445
#>  [91] 1450 1455 1460 1465 1470 1475 1480 1485 1490 1495 1500 1505 1510 1515 1520
#> [106] 1525 1530 1535 1540 1545 1550 1555 1560 1565 1570 1575 1580 1585 1590 1595
#> [121] 1600 1605 1610 1615 1620 1625 1630 1635 1640 1645 1650 1655 1660 1665 1670
#> [136] 1675 1680 1685 1690 1695 1700 1705 1710 1715 1720 1725 1730 1735 1740 1745
#> [151] 1750 1755 1760 1765 1770 1775 1780 1785 1790 1795 1800 1805 1810 1815 1820
#> [166] 1825 1830 1835 1840 1845 1850 1855 1860 1865 1870 1875 1880 1885 1890 1895
#> [181] 1900 1905 1910 1915 1920 1925 1930 1935 1940 1945 1950 1955 1960 1965 1970
#> [196] 1975 1980 1985 1990 1995 2000
# Access the spectra
scratch_OpenSpecy$spectra
#>      runif.n...201.
#>               <num>
#>   1:    0.080750138
#>   2:    0.834333037
#>   3:    0.600760886
#>   4:    0.157208442
#>   5:    0.007399441
#>  ---               
#> 197:    0.906051578
#> 198:    0.772730364
#> 199:    0.383370670
#> 200:    0.999652457
#> 201:    0.349299049
# Access the metadata
scratch_OpenSpecy$metadata
#>        x     y  file_name         col_id                          file_id
#>    <int> <int>     <char>         <char>                           <char>
#> 1:     1     1 fake_noise runif.n...201. 74944ff090b28e2fe0504f0e85d29ded
# Performs checks to ensure that OpenSpecy objects are adhering to our standards;
# returns TRUE if it passes. 
check_OpenSpecy(scratch_OpenSpecy)
#> [1] TRUE

# Checks only the object type to make sure it has OpenSpecy type
is_OpenSpecy(scratch_OpenSpecy)
#> [1] TRUE

We have some generic functions built for inspecting the spectra:

print(scratch_OpenSpecy) # shows the raw object
#>      wavenumber runif.n...201.
#>           <num>          <num>
#>   1:       1000    0.080750138
#>   2:       1005    0.834333037
#>   3:       1010    0.600760886
#>   4:       1015    0.157208442
#>   5:       1020    0.007399441
#>  ---                          
#> 197:       1980    0.906051578
#> 198:       1985    0.772730364
#> 199:       1990    0.383370670
#> 200:       1995    0.999652457
#> 201:       2000    0.349299049
#> 
#> $metadata
#>        x     y  file_name         col_id                          file_id
#>    <int> <int>     <char>         <char>                           <char>
#> 1:     1     1 fake_noise runif.n...201. 74944ff090b28e2fe0504f0e85d29ded
summary(scratch_OpenSpecy) # summarizes the contents of the spectra
#> $wavenumber
#>  Length Min. Max.     Res.
#>     201 1000 2000 4.975124
#> 
#> $spectra
#>  Number Min. Intensity Max. Intensity
#>       1    0.004496308      0.9996525
#> 
#> $metadata
#>   Min. Max.
#> x    1    1
#> y    1    1
#> [1] "x"         "y"         "file_name" "col_id"    "file_id"
head(scratch_OpenSpecy) # shows the top wavenumbers and intensities
#>    wavenumber runif.n...201.
#>         <num>          <num>
#> 1:       1000    0.080750138
#> 2:       1005    0.834333037
#> 3:       1010    0.600760886
#> 4:       1015    0.157208442
#> 5:       1020    0.007399441
#> 6:       1025    0.466393497

Save Data

Open Specy objects can be saved most accurately as .rds, .yml, or .json files. .rds will be the most reproducible as it is a native R file format and floating point errors can happen with .json or .yml.

write_spec(scratch_OpenSpecy, "test_scratch_OpenSpecy.yml", digits = 5)
write_spec(scratch_OpenSpecy, "test_scratch_OpenSpecy.json", digits = 5)

Format Conversions

Another great spectroscopy R package is hyperSpec. We actually depend on their functions for several of ours. They are currently making some awesome new features and we want to integrate well with them so that both packages can be easily used together. That is why we created the as_hyperSpec function and made sure that as_OpenSpecy can convert from hyperSpec objects.

hyperspecy <- as_hyperSpec(scratch_OpenSpecy)

Visualization

Spectra

In R, we have two ways to visualize your spectra, one is quick and efficient and the other is interactive. Here is an example of quick and efficient plotting.

plot(scratch_OpenSpecy) # quick and efficient

This is an example of an interactive plot. You can plot two different datasets simultaneously to compare.

# This will min-max normalize your data even if it isn't already but are not
# changing your underlying data
plotly_spec(scratch_OpenSpecy, json_example)

Maps

Spectral maps can also be visualized as overlaid spectra but in addition the spatial information can be plotted as a heatmap. It is important to note that when multiple spectra are uploaded in batch they are prescribed x and y coordinates, this can be helpful for visualizing summary statistics and navigating vast amounts of data but shouldn’t be confused with data which actually has spatial coordinates.

In R the user can control what values form the colors of the heatmap by specifying z, it is handy to put the information you want in the metadata for this reason. This example just shows the x values of the spectra.

heatmap_spec(spectral_map,
             z = spectral_map$metadata$x)

An interactive plot of the heatmap and spectra overlayed can be generated with the interactive_plot() function. A user can hover over the heatmap to identify the next row id they are interested in and update the value of select to see that spectrum.

interactive_plot(spectral_map, select = 100, z = spectral_map$metadata$x)


Combining OpenSpecy Objects

Sometimes you have several OpenSpecy objects that you want to combine into one. The easiest way to do that is by having spectra which all are in the same exact format with the same series of wavenumbers. The default settings of c_spec assume that is the case.

If you have different wavenumber ranges for the spectra you want to combine, you can set range = "common" and res equal to the wavenumber resolution you want and the function will collapse all the spectra to whatever their common range is using linear interpolation.

c_spec(list(asp_example, ps_example), range = "common", res = 8) |> 
  plot()

Filtering OpenSpecy Objects

OpenSpecy objects can have any number of spectra in them. To create an OpenSpecy with a subset of the spectra that is in an Open Specy object you can use the filter_spec function. Filtering is allowed by index number, name, or using a logical vector. Filtering will update the spectra and metadata items of the OpenSpecy but not the wavenumber.

# Extract the 150th spectrum. 
filter_spec(spectral_map, 150)
#>      wavenumber         9_5
#>           <num>       <num>
#>   1:     717.42  0.06159078
#>   2:     725.13  0.01614512
#>   3:     732.85 -0.02511934
#>   4:     740.56 -0.05653130
#>   5:     748.28 -0.07012609
#>  ---                       
#> 423:    3972.81 -0.23276733
#> 424:    3980.52 -0.22747667
#> 425:    3988.24 -0.23046409
#> 426:    3995.95 -0.23281474
#> 427:    4003.67 -0.22850421
#> 
#> $metadata
#>        y     x       file_name  license
#>    <num> <num>          <char>   <char>
#> 1:     9     5 CA small UF.dat CC BY-NC
#>                                                     description samples lines
#>                                                          <char>   <num> <num>
#> 1: Pixel 15, XPos=12549.72, YPos= 589.82, X=12362.22, Y= 614.82      16    13
#>    bands header offset data type interleave                  z plot titles
#>    <num>         <num>     <num>     <char>                         <char>
#> 1:   427             0         4        bip Wavenumbers (cm-1), Absorbance
#>                  pixel size col_id
#>                      <char> <char>
#> 1: 0.000025000, 0.000025000    9_5
#>                                                           session_id
#>                                                               <char>
#> 1: 4ccaf521fcd9a972e0edd22d7e9615fe/010845f28c4c5dd986f9069350df2845
#>                             file_id
#>                              <char>
#> 1: 215a0fd7e0dc3b1001799214618ce6eb
# Extract the spectrum with column name "8_5". 
filter_spec(spectral_map, "8_5") |> 
  print()
#>      wavenumber         8_5
#>           <num>       <num>
#>   1:     717.42  0.15336494
#>   2:     725.13  0.10891031
#>   3:     732.85  0.06503499
#>   4:     740.56  0.03334329
#>   5:     748.28  0.01523896
#>  ---                       
#> 423:    3972.81 -0.28495014
#> 424:    3980.52 -0.28366613
#> 425:    3988.24 -0.28505880
#> 426:    3995.95 -0.28465354
#> 427:    4003.67 -0.27912781
#> 
#> $metadata
#>        y     x       file_name  license
#>    <num> <num>          <char>   <char>
#> 1:     8     5 CA small UF.dat CC BY-NC
#>                                                     description samples lines
#>                                                          <char>   <num> <num>
#> 1: Pixel 15, XPos=12549.72, YPos= 589.82, X=12362.22, Y= 614.82      16    13
#>    bands header offset data type interleave                  z plot titles
#>    <num>         <num>     <num>     <char>                         <char>
#> 1:   427             0         4        bip Wavenumbers (cm-1), Absorbance
#>                  pixel size col_id
#>                      <char> <char>
#> 1: 0.000025000, 0.000025000    8_5
#>                                                           session_id
#>                                                               <char>
#> 1: 4ccaf521fcd9a972e0edd22d7e9615fe/010845f28c4c5dd986f9069350df2845
#>                             file_id
#>                              <char>
#> 1: 215a0fd7e0dc3b1001799214618ce6eb
# Extract the spectra with a logical argument based on metadata
filter_spec(spectral_map, spectral_map$metadata$y == 1) |>
  plot()

Sampling OpenSpecy Objects

Sometimes you have a large suite of examples of spectra of the same material and you want to reduce the number of spectra you run through the analysis for simplicity or you are running simulations or other procedures that require you to first sample from the spectra contained in your OpenSpecy objects before doing analysis. the sample_spec function serves this purpose.

sample_spec(spectral_map, size = 5) |>
  plot()

Processing

The goal of this all processing is to increase the signal to noise ratio (S/N) of the spectra without distorting the shape, position, or relative size of the peaks. After loading data, you can process the data using intensity adjustment, baseline subtraction, smoothing, flattening, and range selection. The default settings is an absolute derivative transformation, it is kind of magic, it does something similar to smoothing, baseline subtraction, and intensity correction simultaneously and really quickly.

The process_spec() function is a monolithic function for all processing procedures which is optimized by default to result in high signal to noise in most cases, same as the app.

processed <- process_spec(raman_hdpe,
                          active = TRUE,
                          adj_intens = FALSE,
                          adj_intens_args = list(type = "none"),
                          conform_spec = TRUE,
                          conform_spec_args = list(range = NULL, res = 8,
                                                   type = "interp"),
                          restrict_range = FALSE,
                          restrict_range_args = list(min = 0, max = 6000),
                          flatten_range = FALSE,
                          flatten_range_args = list(min = 2200, max = 2420),
                          subtr_baseline = FALSE,
                          subtr_baseline_args = list(type = "polynomial",
                                                     degree = 8, raw = FALSE,
                                                     baseline = NULL),
                          smooth_intens = TRUE,
                          smooth_intens_args = list(polynomial = 3, window = 11,
                                                    derivative = 1, abs = TRUE),
                          make_rel = TRUE)
summary(processed)
#> $wavenumber
#>  Length Min. Max.     Res.
#>     362  304 3192 7.977901
#> 
#> $spectra
#>  Number Min. Intensity Max. Intensity
#>       1              0              1
#> 
#> $metadata
#>   Min. Max.
#> x    1    1
#> y    1    1
#> [1] "x"                 "y"                 "user_name"        
#> [4] "spectrum_type"     "spectrum_identity" "organization"     
#> [7] "license"           "session_id"        "file_id"
summary(raman_hdpe)
#> $wavenumber
#>  Length   Min.    Max.    Res.
#>     964 301.04 3198.12 3.00527
#> 
#> $spectra
#>  Number Min. Intensity Max. Intensity
#>       1             26            816
#> 
#> $metadata
#>   Min. Max.
#> x    1    1
#> y    1    1
#> [1] "x"                 "y"                 "user_name"        
#> [4] "spectrum_type"     "spectrum_identity" "organization"     
#> [7] "license"           "session_id"        "file_id"

You can compare the processed and unprocessed data in an overlay plot.

plotly_spec(raman_hdpe, processed)

We want people to use the process_spec() function for most processing operations. All other processing functions can be tuned using its parameters in the single function and we have set defaults and nested the processing functions in a way that provides typically quality results. However, we recognize that nesting of functions and order of operations can be useful for users to control so you can also use individual functions for each operation if you’d like. See explanations of each processing sub-function below.

Threshold Signal-Noise

Considering whether you have enough signal to analyze spectra is important. Classical spectroscopy would recommend your highest peak to be at least 10 times the baseline of your processed spectra before you begin analysis. If your spectra is below that threshold even after processing, you may want to consider recollecting it. In practice, we are rarely able to collect spectra of that good quality and more often use 5. The “run_sig_over_noise” metric searches your spectra for high and low regions and conducts division on them to derive the signal to noise ratio and step specifies the number of intensities to search. This is a good way to automatically calculate the signal to noise ratio. In the example below you can see that our signal to noise ratio is increased by the processing, the goal of processing is generally to maximize this. “sig_times_noise” metric multiplies the mean signal by the standard deviation of the signal and “tot_sig” metric sums the intensities. The latter can be really useful for thresholding spectral maps to identify particles which we will discuss later. If you know where your signal region and noise regions are you can specify them with sig_min, sig_max, noise_min, and noise_max.

sig_noise(processed, metric = "run_sig_over_noise") > 
  sig_noise(raman_hdpe, metric = "run_sig_over_noise")

If analyzing spectra in batch, we recommend looking at the heatmap and optimizing the percent of spectra that are above your signal to noise threshold to determine the correct settings instead of looking through spectra individually. Setting the min_sn will threshold the heatmap image to only color spectra which have a sn value over the threshold.

spectral_map_p <- spectral_map |>
  process_spec(flatten_range = T)

spectral_map_p$metadata$sig_noise <- sig_noise(spectral_map_p)

heatmap_spec(spectral_map_p, sn = spectral_map_p$metadata$sig_noise, min_sn = 5)

Intensity Adjustment

Open Specy assumes that intensity units are in absorbance units but Open Specy can adjust reflectance or transmittance spectra to absorbance units. The transmittance adjustment uses the \(\log_{10} 1/T\) calculation which does not correct for system or particle characteristics. The reflectance adjustment use the Kubelka-Munk equation \(\frac{(1-R)^2}{2R}\).

This is the respective R code for a scenario where the spectra doesn’t need intensity adjustment:

trans_raman_hdpe <- raman_hdpe
trans_raman_hdpe$spectra <- 2 - trans_raman_hdpe$spectra^2
    
rev_trans_raman_hdpe <- trans_raman_hdpe |> 
  adj_intens(type = "transmittance")

plotly_spec(trans_raman_hdpe, rev_trans_raman_hdpe)

Conforming

Conforming spectra is essential before comparing to a reference library and can be useful for summarizing data when you don’t need it to be highly resolved spectrally. We set the default spectral resolution to 8 because this tends to be pretty good for a lot of applications and is in between 4 and 8 which are commonly used wavenumber resolutions. There are several ways that this function can be specified.

conform_spec(raman_hdpe, res = 8) |> # convert res to 8 wavenumbers.
  summary()
#> $wavenumber
#>  Length Min. Max.     Res.
#>     362  304 3192 7.977901
#> 
#> $spectra
#>  Number Min. Intensity Max. Intensity
#>       1       42.85152       602.4639
#> 
#> $metadata
#>   Min. Max.
#> x    1    1
#> y    1    1
#> [1] "x"                 "y"                 "user_name"        
#> [4] "spectrum_type"     "spectrum_identity" "organization"     
#> [7] "license"           "session_id"        "file_id"

# Force one spectrum to have the exact same wavenumbers as another
conform_spec(asp_example, range = ps_example$wavenumber, res = NULL) |> 
  summary()
#> $wavenumber
#>  Length     Min.     Max.     Res.
#>    1736 651.8728 3998.025 1.927507
#> 
#> $spectra
#>  Number Min. Intensity Max. Intensity
#>       1   0.0009781419      0.5156938
#> 
#> $metadata
#>   Min. Max.
#> x    1    1
#> y    1    1
#> [1] "x"          "y"          "file_name"  "license"    "col_id"    
#> [6] "session_id" "file_id"

# Specify the wavenumber resolution and use a rolling join instead of linear
# approximation (faster for large datasets). 
conform_spec(spectral_map, range = ps_example$wavenumber, res = 10,
             type = "roll") |> 
  summary()
#> $wavenumber
#>  Length Min. Max.     Res.
#>     329  720 4000 9.969605
#> 
#> $spectra
#>  Number Min. Intensity Max. Intensity
#>     208      -1.317072       1.168225
#> 
#> $metadata
#>   Min. Max.
#> x    0   15
#> y    0   12
#>  [1] "y"             "x"             "file_name"     "license"      
#>  [5] "description"   "samples"       "lines"         "bands"        
#>  [9] "header offset" "data type"     "interleave"    "z plot titles"
#> [13] "pixel size"    "col_id"        "session_id"    "file_id"

Smoothing

The Savitzky-Golay filter is used for smoothing. Higher polynomial numbers lead to more wiggly fits and thus less smoothing, lower numbers lead to more smooth fits. The SG filter is fit to a moving window of 11 data points by default where the center point in the window is replaced with the polynomial estimate. Larger windows will produce smoother fits. The derivative order is set to 1 by default which transforms the spectra to their first derivative. A zero order derivative will have no derivative transformation. When smoothing is done well, peak shapes and relative heights should not change. The absolute value is primarily useful for first derivative spectra where the absolute value results in an absorbance-like spectrum which is why we set it as the default. You’ll notice a new function we are using c_spec() which is used to combine spectral objects into one OpenSpecy object.

Examples of smoothing:

none <- make_rel(raman_hdpe)
p1 <- smooth_intens(raman_hdpe, polynomial = 1, derivative = 0, abs = F)
p4 <- smooth_intens(raman_hdpe, polynomial = 4, derivative = 0, abs = F)

c_spec(list(none, p1, p4)) |> 
  plot()
Sample `raman_hdpe` spectrum with different smoothing polynomials.

Sample raman_hdpe spectrum with different smoothing polynomials.

Derivative transformation can happen with the same function.

none <- make_rel(raman_hdpe)
d1 <- smooth_intens(raman_hdpe, derivative = 1, abs = T)
d2 <- smooth_intens(raman_hdpe, derivative = 2, abs = T)

c_spec(list(none, d1, d2)) |> 
  plot()
Sample `raman_hdpe` spectrum with different derivatives.

Sample raman_hdpe spectrum with different derivatives.

Baseline Correction

The goal of baseline correction is to get all non-peak regions of the spectra to zero absorbance. The higher the polynomial order, the more wiggly the fit to the baseline. If the baseline is not very wiggly, a more wiggly fit could remove peaks which is not desired. The baseline correction algorithm used in Open Specy is called “iModPolyfit” (Zhao et al. 2007). This algorithm iteratively fits polynomial equations of the specified order to the whole spectrum. During the first fit iteration, peak regions will often be above the baseline fit. The data in the peak region is removed from the fit to make sure that the baseline is less likely to fit to the peaks. The iterative fitting terminates once the difference between the new and previous fit is small. An example of a good baseline fit below. Manual baseline correction can also be specified by providing a baseline OpenSpecy object.

alternative_baseline <- smooth_intens(raman_hdpe, polynomial = 1, window = 51,
                                      derivative = 0, abs = F, make_rel = F) |>
  flatten_range(min = 2700, max = 3200, make_rel = F)

none <- make_rel(raman_hdpe)
d2 <- subtr_baseline(raman_hdpe, type = "manual",
                     baseline = alternative_baseline)
d8 <- subtr_baseline(raman_hdpe, degree = 8)

c_spec(list(none, d2, d8)) |> 
  plot()
Sample `raman_hdpe` spectrum with different degrees of background subtraction (Cowger et al., 2020).

Sample raman_hdpe spectrum with different degrees of background subtraction (Cowger et al., 2020).

Range Selection

Sometimes an instrument operates with high noise at the ends of the spectrum and, a baseline fit produces distortions, or there are regions of interest for analysis. Range selection accomplishes those goals. You should look into the signal to noise ratio of your specific instrument by wavelength to determine what wavelength ranges to use. Distortions due to baseline fit can be assessed from looking at the process spectra. Additionally, you can restrict the range to examine a single peak or a subset of peaks of interests. Multiple ranges can be specified simultaneously.

none <- make_rel(raman_hdpe)
r1 <- restrict_range(raman_hdpe, min = 1000, max = 2000)
r2 <- restrict_range(raman_hdpe, min = c(1000, 1800), max = c(1200, 2000))

compare_ranges <- c_spec(list(none, r1, r2), range = "common")
# Common argument crops the ranges to the most common range between the spectra
# when joining. 

plot(compare_ranges)
Sample `raman_hdpe` spectrum with different degrees of range restriction.

Sample raman_hdpe spectrum with different degrees of range restriction.

Flattening Ranges

Sometimes there are peaks that really shouldn’t be in your spectra and can distort your interpretation of the spectra but you don’t necessarily want to remove the regions from the analysis because you believe those regions should be flat instead of having a peak. One way to deal with this is to replace the peak values with the mean of the values around the peak. This is the purpose of the flatten_range function. By default it is set to flatten the CO2 region for FTIR spectra because that region often needs to be flattened when atmospheric artifacts occur in spectra. Like restrict_range, the R function can accept multiple ranges.

single <- filter_spec(spectral_map, 120) # Function to filter spectra by index
                                         # number or name or a logical vector. 
none <- make_rel(single)
f1 <- flatten_range(single) #default flattening the CO2 region. 
f2 <- flatten_range(single, min = c(1000, 2500), max = c(1200, 3000))

compare_flats <- c_spec(list(none, f1, f2))

plot(compare_flats)
Sample `raman_hdpe` spectrum with different degrees of background subtraction (Cowger et al., 2020).

Sample raman_hdpe spectrum with different degrees of background subtraction (Cowger et al., 2020).

Min-Max Normalization

Often we regard spectral intensities as arbitrary and min-max normalization allows us to view spectra on the same scale without drastically distorting their shapes or relative peak intensities. In the package, most of the processing functions will min-max transform your spectra by default if you do not specify otherwise. This plot shows two plots that are nearly identical except for the min-max transformed spectrum has a y axis that ranges from 0-1.

relative <- make_rel(raman_hdpe)

Identifying Spectra

Reading Libraries

Reference libraries are spectra with known identities. The Open Specy library now has over 10,000 spectra in it an is getting so large that we cannot fit it within the R package size limit of 5 MB. We host the reference libraries on OSF and have a function to pull the libraries down automatically. Running get_lib by itself will download all libraries to your package director or you can specify which libraries you want and where you want them.

get_lib(type = "derivative")

After download the libraries you can load them into your active environment. You should load them one at a time. Libraries are also OpenSpecy objects so you can use any Open Specy object as a library.

lib <- load_lib(type = "derivative")

Matches

Before attempting to use a reference library to identify spectra it is really important to understand what format the reference library is in. All the OpenSpecy reference libraries are in Absorbance units. derivative has been absolute first derivative transformed, nobaseline has been baseline corrected, raw is the rawest form of the reference spectra (not recommended except for advanced uses). The previously mentioned libraries all have Raman and FTIR spectra in them. mediod is the mediod compressed library version of the absolute first derivative for FTIR, model is an exception because it is a multinomial regression approach for FTIR to identification of absolute derivative spectra. In this example we use the data("test_lib") which is a subsampled version of the absolute derivative library and data("raman_hdpe") which is an unprocessed Raman spectrum in absorbance units of HDPE plastic. With single spectra it is easy to look at the spectra but when doing in batch, also refer to the Thresholding Signal-Noise section for guidance on making sure your batch spectra are processed to quality specs.

data("test_lib")
data("raman_hdpe")

processed <- process_spec(x = raman_hdpe, 
                          conform_spec = F, #We will conform during matching. 
                          smooth_intens = T #Conducts the default derivative transformation. 
                          )

# Check to make sure that the signal to noise ratio of the processed spectra is
# greater than 10.
print(sig_noise(processed) > 10)
plotly_spec(raman_hdpe, processed)

After your spectra is processed similarly to the library specifications, you can identify the spectra using match_spec(). All of the libraries have wavenumbers at 8 cm^-1 resolution. Whichever library you choose, you need to get your spectra into a similar enough format to use for comparison. That means conforming the wavenumbers to the same values using conform = T. Alternatively, you could have done the conformation during the processing. The add_library_metadata and add_object_metadata options specify the column name in the metadata that you want to add metadata from and top_n specifies how many matches you want. In this example we just identified a single spectrum with the library but you can also send an OpenSpecy object with multiple spectra. The output matches is a data.table with at least 3 columns, object_id tells you the column names of the spectra in x, library_id tells you the column names from the library that it matched to. match_val is the value of the Pearson correlation coefficient (default) or other correlation if specified in ... or if using the model identification option match_val will be the model confidence. The output in this example returned the correct material type, HDPE, as the top match. If using Pearson correlation, 0.7 is a good threshold to use for a positive ID. In this example, only our top match is greater than the threshold so we would disregard the other matches. If no matches were above our threshold, we would proclaim that the spectrum is of an unknown identity. You’ll also notice in this example that we matched to a library with both Raman and FTIR spectra but the Raman spectra had the highest hits, this is the rationale for lazily matching to a library with both. If you want to just match to a library with FTIR or Raman spectra, you can first filter the library using filter_spec() using SpectrumType.

matches <- match_spec(x = processed, library = test_lib, conform = T,
                      add_library_metadata = "sample_name", top_n = 5)
print(matches[,c("object_id", "library_id", "match_val", "SpectrumType",
                 "SpectrumIdentity")])

Library Metadata

The libraries we have created have over 100 variables of metadata in them and this can be onerous to read through especially given that many of the variables are NA values. We created get_metadata() to remedy this by removing columns from the metadata which are all blank values. The function below will return the metadata for the top match in matches. Remember, similar filter_spec(), you can specify logic for more than one thing at a time.

get_metadata(x = test_lib, logic = matches[[1,"library_id"]], rm_empty = T)

Plot Matches

Overlaying unknown spectra and the best matches can be extremely useful to identify peaks that don’t fit to the reference library which may need further investigation. The example below shows great correspondence between the best match and the unknown spectrum. All major peaks are accounted for and the correct relative height. There are two small peaks in the unknown spectrum near 500 that are not accounted for which could be investigated further but we would call this a positive id to HDPE.

plotly_spec(processed, filter_spec(test_lib, logic = matches[[1,"library_id"]]))

Sharing Reference Data

If you have reference data or AI models that you think would be useful for sharing with the spectroscopy community through OpenSpecy please contact the package administrator to discuss options for collaborating.

Characterizing Particles

Sometimes the spectroscopy task we want to perform is to identify particles in a spectral map. This is especially common for microplastic analysis where a spectral map is used to image a sample and spectral information is used to differentiate microplastic particles from nonplastic particles. In addition to the material id, one often wants to measure the shape and size of the particles. In a brute force technique, one could first identify every spectrum in the map, then use thresholding and image analysis to measure the particles. However, more often than not, particles are well separated on the image surface and background spectra is quite different from particle spectra and therefore we can use thresholding a priori to identify and measure the particles, then pass an exemplary spectrum for each particle to the identification routine. It is important to note here that this is at the bleeding edge of theory and technique so we may be updating these functions in the near future.

Brute Force

data("test_lib")
test_map <- read_any(read_extdata("CA_tiny_map.zip"))

test_map_processed <- process_spec(test_map, conform_spec_args = list(
  range = test_lib$wavenumber, res = NULL)
  )

identities <- match_spec(test_map_processed, test_lib, order = test_map,
                         add_library_metadata = "sample_name", top_n = 1)

features <- ifelse(identities$match_val > 0.7,
                   tolower(identities$polymer_class), "unknown")

id_map <- def_features(x = test_map_processed, features = features)

id_map$metadata$identities <- features
# Also should probably be implemented automatically in the function when a
# character value is provided. 

# Collapses spectra to their median for each particle
test_collapsed <- collapse_spec(id_map)

A Priori Particle Thresholding

data("test_lib")
test_map <- read_any(read_extdata("CA_tiny_map.zip"))

# Characterize the total signal as a threshold value. 
snr <- sig_noise(test_map,metric = "log_tot_sig")

# Use this to find your particles and the sig_noise value to use for
# thresholding. 
heatmap_spec(test_map, z = snr)

# Set define the feature regions based on the threshold. 400 appeared to be
# where I suspected my particle to be in the previous heatmap. 
id_map <- def_features(x = test_map, features = snr > 400) 

# Check that the thresholding worked as expected.
heatmap_spec(id_map, z = id_map$metadata$feature_id)

# Collapse the spectra to their medians based on the threshold. Important to
# note here that the particles with id -88 are anything from the FALSE values
# so they should be your background. 
collapsed_id_map <- id_map |>
  collapse_spec()

# Process the collapsed spectra. 
id_map_processed <- process_spec(collapsed_id_map, conform_spec_args = list(
  range = test_lib$wavenumber, res = NULL)
  )

# Check the spectra.
plot(id_map_processed)

# Get the matches of the collapsed spectra for the particles.
matches <- match_spec(id_map_processed, test_lib,
                      add_library_metadata = "sample_name", top_n = 1)

References

Chabuka BK, Kalivas JH (2020). “Application of a Hybrid Fusion Classification Process for Identification of Microplastics Based on Fourier Transform Infrared Spectroscopy.” Applied Spectroscopy, 74(9), 1167–1183. doi: 10.1177/0003702820923993.

Cowger W, Gray A, Christiansen SH, De Frond H, Deshpande AD, Hemabessiere L, Lee E, Mill L, et al. (2020). “Critical Review of Processing and Classification Techniques for Images and Spectra in Microplastic Research.” Applied Spectroscopy, 74(9), 989–1010. doi: 10.1177/0003702820929064.

Cowger W, Steinmetz Z, Gray A, Munno K, Lynch J, Hapich H, Primpke S, De Frond H, Rochman C, Herodotou O (2021). “Microplastic Spectral Classification Needs an Open Source Community: Open Specy to the Rescue!” Analytical Chemistry, 93(21), 7543–7548. doi: 10.1021/acs.analchem.1c00123.

Primpke S, Wirth M, Lorenz C, Gerdts G (2018). “Reference Database Design for the Automated Analysis of Microplastic Samples Based on Fourier Transform Infrared (FTIR) Spectroscopy.” Analytical and Bioanalytical Chemistry, 410(21), 5131–5141. doi: 10.1007/s00216-018-1156-x.

Renner G, Schmidt TC, Schram J (2017). “A New Chemometric Approach for Automatic Identification of Microplastics from Environmental Compartments Based on FT-IR Spectroscopy.” Analytical Chemistry, 89(22), 12045–12053. doi: 10.1021/acs.analchem.7b02472.

Savitzky A, Golay MJ (1964). “Smoothing and Differentiation of Data by Simplified Least Squares Procedures.” Analytical Chemistry, 36(8), 1627–1639.

Zhao J, Lui H, McLean DI, Zeng H (2007). “Automated Autofluorescence Background Subtraction Algorithm for Biomedical Raman Spectroscopy.” Applied Spectroscopy, 61(11), 1225–1232. doi: 10.1366/000370207782597003.