After the dismal failure of luminescent infectivity quantification, and the lack of success in infecting phage due to the small sample volumes in use and the wrong phage type. The plaque assay took too long.
+
"The excited state lifetime of PG in buffer is very short, \(4 \pm 3 \text{ps}\), but in complex with DNA it increases almost 1000-fold, reaching a value of \(4.4 \pm 0.01 \text{ns}\)."
No provision for magnetic shielding of the PMT was made.
As with many other experiments in this project, many negative results reported were tainted by the use of such a low sample volume.
The typical method to detect. A quantitative PCR
Such a device is known in biology as a plate reader.
Nanodrop, using 280 nm absorbance. They’re also $10,000.
-
Contrary to luminescence, you have control over when the excitation and emission light turns on. This doesn’t subtract effects like the excitation light from filter leakage
+
A crucial advantage. Contrary to luminescence: you have control over when the excitation and emission light turns on.
+
Error in luminescence can occur due to variation in detector "dark counts" (a problem that plagued luminescence tests),
+
Error in fluorescence
+
This doesn’t subtract effects like the excitation light from filter leakage
Conveniently, T4r has an extraordinarily large genome of approximately 172 kBp dsDNA(Yap and Rossmann 2014); each virion therefore For comparison, a fingerprint has between 0.042 and 0.14 ng of DNA (Subhani, Daniel, and Frascione 2019).
-
While these quantities are small, it is not particularly challenging, and it is not our intention to suggest that this is a good design; we are simply reporting on Designs for systems with comparable performance are . The similar performance despite extremely high detector sensitivity is probably due to the small light-collecting area due to the objective, and
+
While these quantities may sound small, they are not particularly challenging to detect, and it is not our intention to suggest that this is a good design that should be replicated. We are simply reporting on the system that was found to be functional. Designs for systems with comparable performance and even simpler are reported. Despite the extremely high detector sensitivity introduced by photon-counting, similar sensitivity is found. This is probably due to the small light-collecting area due to the objective, and
We show that both fluorescence and the excited state lifetime of SG dramatically increase in viscous solvents, demonstrating an approximate 200-fold enhancement in 100 % glycerol, compared to water, which also makes SG a prospective fluorescent viscosity probe.
-
Biotium GelGreen has a very specific advantage: to increase the safety of the dye, the flurophore is tied to some huge proprietary molecule, preventing it from diffusing through membranes or capsids. This has the side effect of making the fluorescence intensity strictly related to the quantity of genomic material dispersed in the solvent, rather than
-
Luckily, a recent paper has the answer: direct fluorescent detection of DNA in solution, outside using dyes that bind to (intercalate into) DNA. GelGreen doesn’t penetrate.
-
A similar method (using fluroescence microscopy rather than photon counting) was also used by, and is generally a common practice in the bioeffects field.
Biotium GelGreen has a very specific advantage for this specific application. To increase the safety of the dye, two flurophore monomers have been connected into a dimer with a long backbone "bridge", all but preventing it from diffusing through membranes or capsids.
+
+
"On average, these dimeric dyes have a molecular weight at least 2-3 times that of SYBR Safe or SYBR Green I and bear two positive charges as opposed to only one positive charge for SYBR Safe, for example. The much larger sizes as well as the higher charge of GelRed and GelGreen render them difficult to cross the cell membranes, thus denying the opportunity for the dyes to interfere with any intracellular activities, including activities associated with genomic DNA. Consequently, GelRed and GelGreen are not only nonmutagenic but also noncytotoxic within the dye concentration range normally used for nucleic acid gel staining. Furthermore, dimeric dyes such as GelRed and GelGreen exhibit exceptional signal-to-noise ratio because the dyes self-quench in the absence of nucleic acids to result in very low background fluorescence."
This has the side effect of making the fluorescence intensity strictly related to the quantity of genomic material dispersed in the solvent, not within intact virions.
+
It is a shame to
+
I was not able to find information on the thermal degradation of these dyes, in case it was desired tot inactivate after adding the fluorophore.
+
A similar method (using fluroescence microscopy rather than fluorometry and photon counting) was also used by (Madiyar et al. 2017), and is generally a common practice in the bioeffects field.
Somewhat more challenging than viewing PCR output on a gel, since the total quantity of DNA involved is quite low
extra credit: how many photons are released?
Unlike luminescence techniques, lock-in is possible
-
Xu et al use
-
Gel-Doc
Flurophore
+
Luckily, a recent paper has the answer: direct fluorescent detection of DNA in solution, outside using dyes that bind to (intercalate into) DNA.
the prototypical stain is Ethidium Bromide, but is challenging to obtain outside certain laboratories. GelGreen is safe, very stable against photobleaching and long-term storage, inexpensive, and readily available. GelGreen is an Acridine orange (N-alkylacridinium) dye with a similar spectra to green fluorescent protein.
the base had integral overcurrent protection, which was triggered a few times during development - a very useful
For one thing, GelGreen appears to be eminiently stable - samples can be stored for long periods of time, pre-mixed batches.
bleaching was not obviously an issue. A calibration sample was stored in a dark area with the dye bound to DNA for several months with less than 4% decay observed1.
-
tom Lexan cuvettes unexpectedly overwhelmed the DNA signal A surplus Hammamatsu R4220 with HC123 current-limiting base at maximum sensitivity was used. A low-voltage silicon photomultiplier like ON Semi’s C-Series SiPMs will probably be sufficient in most cases.
+
A surplus Hammamatsu R4220 with HC123 current-limiting base at maximum sensitivity was used. A low-voltage silicon photomultiplier like ON Semi’s C-Series SiPMs will probably be sufficient in most cases. Unlike avalanche photodetectors, SPADs and SiPMs have similar gain properties to PMTs,
(Phi6 uses an RNA - many dyes have different responses to single-stranded (ss)DNA, dsDNA, or
As noted by [xi?], this is a saturating effect; if too many flurophores intercalate into the DNA the fluorescence is weaker.
GelGreen is also sensitive to ssDNA and ssRNA but with 5 times lower efficiency. GelGreen absorbs maximally at 272 nm and 507 nm and emits maximally at 528 nm.(Biotium Inc. n.d.)
@@ -258,11 +264,8 @@
Flurophore
Biotium GelRed/GelGreen fluorescence spectra. Credit Biotium Inc, reproduced without permission.
Notably, contrary to most arrangments, the excitation light was input through the transparent bottom of the sample holders. They report that "the limiting factor is the [filter leakage; in their paper they refer to this as noise, distinct from image sensor noise]".
-
+
Notably, contrary to most arrangements, the excitation light was input through the transparent bottom of the sample holders. They report that "the limiting factor is the [filter leakage; in their paper they refer to this as noise, distinct from image sensor noise]".
An incidental advantage is that a color filter makes it easy to diagnose issues with the light path. It is clear whether background,
Lesson learned: color feedback
@@ -300,12 +303,12 @@
Cuvette
Light source
Argon-gas lasers emit several closely-spaced lines in the visible spectrum, the most prominent of which is at 488 nm.
-
Cyan diode lasers emitting at 488 nm are SHARP GH04850B2G appears to be obsolete.
+
Cyan diode lasers emitting at 488 nm can be found on surplus markets. Modules using SHARP GH04850B2G diode are available, although this appears to be obsolete.
Due to their coherent emission, eye protection or suitable interlocks are important when working with lasers. (Are tightly filtered LEDs coherent?)
Various impromptu tests were made with a 2.5W 445 nm laser cutter. Commodity blue 445 nm emitters only barely clip the absorption spectrum of GelGreen, apparently leading to a poor fluorescence yield. Combined with the very high intensity, an unusably high background was found even with the large 0.4 mL test sample. This observation were tainted by a very poor optical arrangement and was largely inconclusive; 445 nm laser diode alone was never tested with the final optical arrangement, and may well have provided sufficient. This may allow the elimination of the excitation filter.
+
Various impromptu tests were made with a 2.5W 445 nm laser cutter. Commodity blue 445 nm emitters only barely clip the absorption spectrum of GelGreen, leading to a much lower fluorescence signal and signal-to-background than higher wavelengths. These observations were tainted by a very poor optical arrangement and were largely inconclusive; the 445 nm laser diode alone was never tested with the final optical arrangement, and may well have proved sufficient. This may allow the elimination of the excitation filter.
(Note that inexpensive laser cutter modules are often intensity modulated via PWM rather than via analog current; this can cause great confusion if not accounted for).
gel doc papers use leds without filters, and paper says narrow leds exist, talk about results with leds
Blue LEDs [Cree XLamp XP-E2 Blue Starboard] are then sufficient for excitation (though high CRI white LEDs emit more 480 nm blue, green leakage is too high).
@@ -339,6 +342,7 @@
Excitation filter used
(486 nm is the \(n=4\) Balmer line for hydrogen).
(note; this was a limited-stock clearance item that has since been discontinued. Equivalent filters are readily available from other suppliers, such as the ThorLabs FL05488-10 or Newport 05LF10-488).
The anti-reflective side faced the LED; the reflective side faced the fiber optic.
+
This does mean is filtered before entering the fiber optic. If some component of the fiber optic assembly were fluorescent, this would contribute to a background.
ThorLabs FGB7 emission filter superficially looks satisfactory, but the tails are believed to be too long to be useable.
Emission filters used
Two filters in series, one:
@@ -351,7 +355,6 @@
Emission filters used
The 10 nm FWHM emission filter is much narrower than professional GFP filters, especially the super-wide edge-pass ones; a lot of photons are lost that way, decreasing efficiency. However, it still appears to be more than sufficient.
manuals for gel-docs typically suggest #16 or #15. SYBR recommends #15.
For an even lower-cost system, orange or amber acrylic sheets (typically used for UV filtering) with similar filtering spectra (e.g. Acrylite 408-5) also exist; such a filter is used on the Carolina gel-doc, for instance.
-
This does mean is filtered before entering the fiber optic.
@@ -362,13 +365,14 @@
Gel filter
Unlike gels or plastic-glass sandwiches, thin-film filters have the property that the pass-band depends greatly on the angle of incidence as \[\lambda_{\text{shifted}} = \lambda \sqrt{1-\sin(\theta)^2}\]
The wavelength shifts shorter (bluer) as \(\theta\) increases. This can be a helpful property for creating tunable filters, but is a nuisance here. This isn’t an issue for the excitation filter, but is an issue for the.
Thin-film dielectric filters also age; ThorLabs considers filters to have a lifetime of 2 years.
+
Noncollimated light, the the band of a dielectric interference bandpass filter shifts = \(488 * (sqrt(1-(sin(45 deg)/2.08)^2)) = 458\); it only shortens in wavelength. This would be a problem for the emission filter, except we’re using a colored-glass filter for that side.
Since the LEDs and flurophore emissions are not naturally collimated, this poses a little bit of an issue. The microscope objective seems to provide sufficient collimation for the emission filter.
The Edmund filter was unmounted glass. Also, these thin-film filters do not extend to the edge of the glass. Edge-blackening ("inked") filters
-
The wavelength can also be shifted by a few nm over the temperature range 0 to 50
+
The wavelength can also be shifted by a few nm over temperature.
The LED was about 3 cm away, and put through a 3 mm aperture in a piece of PVC pipe. The output from the filter was directly into the 1 mm fiber, itself improving the bandpass.
Electronics
-
Monitoring the output with a 5, a 100 ns switching time was typical. A modulation frequency of 1 MHz was achievable, but no
+
Monitoring the output with a photodiode 5, a 100 ns switching time was typical. A modulation frequency of 1 MHz was achievable, but no
While drift in the power supply does not affect the background lock-in, it can affect run-to-run repeatability.
Running FPGA designs at varying speeds helps to debug race-condition related bugs. 6
@@ -419,16 +423,11 @@
Time-domain or time-corr
Polarization is a neat way to filter; linearly polarize the excitation, the emission comes out whatever orientation the DNA happens to be, which is usually random. Apparently
Performance and characteristics
Performance of this arrangement was very satisfactory. A 10-second integration time, with, produced a background fluorescence signal of 1500 counts, with per-sample stability of approximately \(\pm 1500\) counts. The
Arecchi, F. T., E. Gatti, and A. Sona. 1966. “Measurement of Low Light Intensities by Synchronous Single Photon Counting.”Review of Scientific Instruments 37 (7): 942–48. https://doi.org/10.1063/1.1720370.
-
-Balsam, Joshua, Hugh Alan Bruck, Yordan Kostov, and Avraham Rasooly. 2012. “Image Stacking Approach to Increase Sensitivity of Fluorescence Detection Using a Low Cost Complementary Metal-Oxide-Semiconductor (CMOS) Webcam.”Sensors and Actuators B: Chemical 171–172 (August): 141–47. https://doi.org/10.1016/j.snb.2012.02.003.
-
+Madiyar, Foram Ranjeet, Sherry L. Haller, Omer Farooq, Stefan Rothenburg, Christopher Culbertson, and Jun Li. 2017. “AC Dielectrophoretic Manipulation and Electroporation of Vaccinia Virus Using Carbon Nanoelectrode Arrays.”ELECTROPHORESIS 38 (11): 1515–25. https://doi.org/10.1002/elps.201600436.
+
-Xu, Jian, Steven H. DeVries, and Yongling Zhu. 2020. “Quantification of Adeno-Associated Virus with Safe Nucleic Acid Dyes.”bioRxiv, February, 2020.02.27.968636. https://doi.org/10.1101/2020.02.27.968636.
-
Yap, Moh Lan, and Michael G Rossmann. 2014. “Structure and Function of Bacteriophage T4.”Future Microbiology 9 (October): 1319–27. https://doi.org/10.2217/fmb.14.91.
diff --git a/README.md b/README.md
index bf4d7cd..a8e30b2 100644
--- a/README.md
+++ b/README.md
@@ -5,13 +5,14 @@ bibliography: ../../documents/references.bib
- [Amplification-free fluorescent nucleic acid detection via
synchronous photon
counting](#amplification-free-fluorescent-nucleic-acid-detection-via-synchronous-photon-counting)
+- [This document currently lives at
+ GitHub.](#this-document-currently-lives-at-github.)
- [Executive summary](#executive-summary)
- [Initial state](#initial-state)
- [Review](#review)
- [Flurophore](#flurophore)
- [Simple CMOS image stacking
detection](#simple-cmos-image-stacking-detection)
- - [[@Image2012]](#section)
- [Photomultiplier photon wavelength
discrimination](#photomultiplier-photon-wavelength-discrimination)
- [Possible artifacts and deficiencies in
@@ -30,12 +31,15 @@ bibliography: ../../documents/references.bib
- [Performance and
characteristics](#performance-and-characteristics)
- [Literature review](#literature-review)
- - [[@Instrument1957]](#section-1)
# Amplification-free fluorescent nucleic acid detection via synchronous photon counting
+# [This document currently lives at GitHub.](https://github.com/0xDBFB7/fluorescence_photon_counting/releases/download/v0.01/fluorescence.pdf)
+
{width="\\textwidth"}
+Please pardon the crudity of assembly.
+
{width="\\textwidth"}
{width="\\textwidth"}
@@ -58,6 +62,10 @@ After the dismal failure of luminescent infectivity quantification, and
the lack of success in infecting phage due to the small sample volumes
in use and the wrong phage type. The plaque assay took too long.
+\"The excited state lifetime of PG in buffer is very short,
+$4 \pm 3 \text{ps}$, but in complex with DNA it increases almost
+1000-fold, reaching a value of $4.4 \pm 0.01 \text{ns}$.\"
+
No provision for magnetic shielding of the PMT was made.
As with many other experiments in this project, many negative results
@@ -69,49 +77,75 @@ Such a device is known in biology as a plate reader.
Nanodrop, using 280 nm absorbance. They're also \$10,000.
-Contrary to luminescence, you have control over when the excitation and
-emission light turns on. This doesn't subtract effects like the
-excitation light from filter leakage
+A crucial advantage. Contrary to luminescence: you have control over
+when the excitation and emission light turns on.
+
+Error in luminescence can occur due to variation in detector \"dark
+counts\" (a problem that plagued luminescence tests),
+
+Error in fluorescence
+
+This doesn't subtract effects like the excitation light from filter
+leakage
Conveniently, T4r has an extraordinarily large genome of approximately
172 kBp dsDNA[@Structure2014]; each virion therefore For comparison, a
fingerprint has between 0.042 and 0.14 ng of DNA [@DNA2019].
-While these quantities are small, it is not particularly challenging,
-and it is not our intention to suggest that this is a good design; we
-are simply reporting on Designs for systems with comparable performance
-are . The similar performance despite extremely high detector
-sensitivity is probably due to the small light-collecting area due to
-the objective, and
+While these quantities may sound small, they are not particularly
+challenging to detect, and it is not our intention to suggest that this
+is a good design that should be replicated. We are simply reporting on
+the system that was found to be functional. Designs for systems with
+comparable performance and even simpler are reported. Despite the
+extremely high detector sensitivity introduced by photon-counting,
+similar sensitivity is found. This is probably due to the small
+light-collecting area due to the objective, and
## Review
-[@Quantification2020] quantifies adenovirus titer with a ssDNA 4.7 kbase
-genome.
+quantifies adenovirus titer with a ssDNA 4.7 kbase genome.
With a GelRed dye and 528/20 (note: BioTek filters are specified as
center wavelength / FWHM).
-[@SYBR2012] [@Characterization2010] offer excellent
+[@SYBR2012] offer excellent
-We show that both fluorescence and the excited state lifetime of SG
-dramatically increase in viscous solvents, demonstrating an approximate
-200-fold enhancement in 100 % glycerol, compared to water, which also
-makes SG a prospective fluorescent viscosity probe.
+[@Characterization2010]
-Biotium GelGreen has a very specific advantage: to increase the safety
-of the dye, the flurophore is tied to some huge proprietary molecule,
-preventing it from diffusing through membranes or capsids. This has the
-side effect of making the fluorescence intensity strictly related to the
-quantity of genomic material dispersed in the solvent, rather than
+Biotium GelGreen has a very specific advantage for this specific
+application. To increase the safety of the dye, two flurophore monomers
+have been connected into a dimer with a long backbone \"bridge\", all
+but preventing it from diffusing through membranes or capsids.
-Luckily, a recent paper has the answer: direct fluorescent detection of
-DNA in solution, outside using dyes that bind to (intercalate into) DNA.
-GelGreen doesn't penetrate.
+> \"On average, these dimeric dyes have a molecular weight at least 2-3
+> times that of SYBR Safe or SYBR Green I and bear two positive charges
+> as opposed to only one positive charge for SYBR Safe, for example. The
+> much larger sizes as well as the higher charge of GelRed and GelGreen
+> render them difficult to cross the cell membranes, thus denying the
+> opportunity for the dyes to interfere with any intracellular
+> activities, including activities associated with genomic DNA.
+> Consequently, GelRed and GelGreen are not only nonmutagenic but also
+> noncytotoxic within the dye concentration range normally used for
+> nucleic acid gel staining. Furthermore, dimeric dyes such as GelRed
+> and GelGreen exhibit exceptional signal-to-noise ratio because the
+> dyes self-quench in the absence of nucleic acids to result in very low
+> background fluorescence.\"
+
+[@Methods2014]
+
+This has the side effect of making the fluorescence intensity strictly
+related to the quantity of genomic material dispersed in the solvent,
+not within intact virions.
+
+It is a shame to
-A similar method (using fluroescence microscopy rather than photon
-counting) was also used by, and is generally a common practice in the
-bioeffects field.
+I was not able to find information on the thermal degradation of these
+dyes, in case it was desired tot inactivate after adding the
+fluorophore.
+
+A similar method (using fluroescence microscopy rather than fluorometry
+and photon counting) was also used by [@AC2017], and is generally a
+common practice in the bioeffects field.
Somewhat more challenging than viewing PCR output on a gel, since the
total quantity of DNA involved is quite low
@@ -122,12 +156,11 @@ extra credit: how many photons are released?
Unlike luminescence techniques, lock-in is possible
-Xu et al use
-
-Gel-Doc
-
## Flurophore
+Luckily, a recent paper has the answer: direct fluorescent detection of
+DNA in solution, outside using dyes that bind to (intercalate into) DNA.
+
the prototypical stain is Ethidium Bromide, but is challenging to obtain
outside certain laboratories. GelGreen is safe, very stable against
photobleaching and long-term storage, inexpensive, and readily
@@ -144,10 +177,11 @@ bleaching was not obviously an issue. A calibration sample was stored in
a dark area with the dye bound to DNA for several months with less than
4% decay observed[^1].
-tom Lexan cuvettes unexpectedly overwhelmed the DNA signal A surplus
-Hammamatsu R4220 with HC123 current-limiting base at maximum sensitivity
-was used. A low-voltage silicon photomultiplier like ON Semi's C-Series
-SiPMs will probably be sufficient in most cases.
+A surplus Hammamatsu R4220 with HC123 current-limiting base at maximum
+sensitivity was used. A low-voltage silicon photomultiplier like ON
+Semi's C-Series SiPMs will probably be sufficient in most cases. Unlike
+avalanche photodetectors, SPADs and SiPMs have similar gain properties
+to PMTs,
(Phi6 uses an RNA - many dyes have different responses to
single-stranded (ss)DNA, dsDNA, or
@@ -164,16 +198,12 @@ reproduced without permission.](gelred_gelgreen){width="50%"}
## Simple CMOS image stacking detection
-::: @empty
-### [@Image2012] {#section}
-
florescein An f1.2 lens is used.
-Notably, contrary to most arrangments, the excitation light was input
+Notably, contrary to most arrangements, the excitation light was input
through the transparent bottom of the sample holders. They report that
\"the limiting factor is the \[filter leakage; in their paper they refer
to this as noise, distinct from image sensor noise\]\".
-:::
An incidental advantage is that a color filter makes it easy to diagnose
issues with the light path. It is clear whether background,
@@ -282,8 +312,9 @@ Lesson learned: beware autofluorescence
Argon-gas lasers emit several closely-spaced lines in the visible
spectrum, the most prominent of which is at 488 nm.
-Cyan diode lasers emitting at 488 nm are SHARP GH04850B2G appears to be
-obsolete.
+Cyan diode lasers emitting at 488 nm can be found on surplus markets.
+Modules using SHARP GH04850B2G diode are available, although this
+appears to be obsolete.
Due to their coherent emission, eye protection or suitable interlocks
are important when working with lasers. (Are tightly filtered LEDs
@@ -297,13 +328,12 @@ lasers are available.
Various impromptu tests were made with a 2.5W 445 nm laser cutter.
Commodity blue 445 nm emitters only barely clip the absorption spectrum
-of GelGreen, apparently leading to a poor fluorescence yield. Combined
-with the very high intensity, an unusably high background was found even
-with the large 0.4 mL test sample. This observation were tainted by a
-very poor optical arrangement and was largely inconclusive; 445 nm laser
-diode alone was never tested with the final optical arrangement, and may
-well have provided sufficient. This may allow the elimination of the
-excitation filter.
+of GelGreen, leading to a much lower fluorescence signal and
+signal-to-background than higher wavelengths. These observations were
+tainted by a very poor optical arrangement and were largely
+inconclusive; the 445 nm laser diode alone was never tested with the
+final optical arrangement, and may well have proved sufficient. This may
+allow the elimination of the excitation filter.
(Note that inexpensive laser cutter modules are often intensity
modulated via PWM rather than via analog current; this can cause great
@@ -413,6 +443,10 @@ suppliers, such as the ThorLabs FL05488-10 or Newport 05LF10-488).
The anti-reflective side faced the LED; the reflective side faced the
fiber optic.
+This does mean is filtered before entering the fiber optic. If some
+component of the fiber optic assembly were fluorescent, this would
+contribute to a background.
+
ThorLabs FGB7 emission filter superficially looks satisfactory, but the
tails are believed to be too long to be useable.
@@ -448,8 +482,6 @@ used for UV filtering) with similar filtering spectra (e.g. Acrylite
408-5) also exist; such a filter is used on the Carolina gel-doc, for
instance.
-This does mean is filtered before entering the fiber optic.
-
::: center
:::
@@ -472,6 +504,11 @@ This isn't an issue for the excitation filter, but is an issue for the.
Thin-film dielectric filters also age; ThorLabs considers filters to
have a lifetime of 2 years.
+Noncollimated light, the the band of a dielectric interference bandpass
+filter shifts = $488 * (sqrt(1-(sin(45 deg)/2.08)^2)) = 458$; it only
+shortens in wavelength. This would be a problem for the emission filter,
+except we're using a colored-glass filter for that side.
+
Since the LEDs and flurophore emissions are not naturally collimated,
this poses a little bit of an issue. The microscope objective seems to
provide sufficient collimation for the emission filter.
@@ -479,8 +516,7 @@ provide sufficient collimation for the emission filter.
The Edmund filter was unmounted glass. Also, these thin-film filters do
not extend to the edge of the glass. Edge-blackening (\"inked\") filters
-The wavelength can also be shifted by a few nm over the temperature
-range 0 to 50
+The wavelength can also be shifted by a few nm over temperature.
The LED was about 3 cm away, and put through a 3 mm aperture in a piece
of PVC pipe. The output from the filter was directly into the 1 mm
@@ -490,8 +526,8 @@ fiber, itself improving the bandpass.
{width="\\textwidth"}
-Monitoring the output with a [^5], a 100 ns switching time was typical.
-A modulation frequency of 1 MHz was achievable, but no
+Monitoring the output with a photodiode [^5], a 100 ns switching time
+was typical. A modulation frequency of 1 MHz was achievable, but no
While drift in the power supply does not affect the background lock-in,
it can affect run-to-run repeatability.
@@ -657,11 +693,7 @@ integration time, with, produced a background fluorescence signal of
1500 counts, with per-sample stability of approximately $\pm 1500$
counts. The
-## Literature review
-
-### [@Instrument1957] {#section-1}
-
-::: #refs :::
+## Literature review {#literature-review .unnumbered}
[^1]: pulse_1.pnw line 2567
diff --git a/generate.sh b/generate.sh
index eadbe00..000dd31 100644
--- a/generate.sh
+++ b/generate.sh
@@ -1 +1,2 @@
-pandoc --toc --bibliography=../../documents/references.bib -t html -s ../../documents/fluorescence.tex -o README.html --citeproc --mathjax --metadata title="a" --metadata link-citations=true
\ No newline at end of file
+pandoc --toc --bibliography=../../documents/references.bib -t html -s ../../documents/fluorescence.tex -o README.html --citeproc --mathjax --metadata title="a" --metadata link-citations=true
+pandoc --toc --bibliography=../../documents/references.bib -t markdown -s ../../documents/fluorescence.tex -o README.md --citeproc