From fa499394fdad953c06db550d5ee4093d27fccd4a Mon Sep 17 00:00:00 2001
From: BasedOnTechnology <therobotist@gmail.com>
Date: Wed, 27 Oct 2021 11:18:56 -0400
Subject: [PATCH] Moved again

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 .gitignore  |   3 +-
 README.md   | 637 ++++++++++++++++++++++++++++++++++++++++++++++++++++
 generate.sh |   3 +-
 3 files changed, 641 insertions(+), 2 deletions(-)
 create mode 100644 README.md

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+---
+bibliography: ../../documents/references.bib
+---
+
+Amplification-free fluorescent nucleic acid detection via synchronous photon counting
+=====================================================================================
+
+![image](fluorescence/fluro_1){width="\\textwidth"}
+
+![Pardon the mess.](fluorescence/fluro_2){width="\\textwidth"}
+
+![image](fluorescence/light_source){width="\\textwidth"}
+
+![image](fluorescence/fiber_optic){width="\\textwidth"}
+
+![image](fluorescence/comparator){width="\\textwidth"}
+
+Quartus Prime 18.1 Lite edition
+
+Executive summary
+-----------------
+
+Cuvette should be opaque or white to avoid autofluro;
+
+Initial state
+-------------
+
+real-time
+
+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.
+
+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
+
+Conveniently, T4r has an extraordinarily large genome of approximately
+172 kBp dsDNA(); each virion therefore For comparison, a fingerprint has
+between 0.042 and 0.14 ng of DNA ().
+
+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
+
+Review
+------
+
+() 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).
+
+() () 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.
+
+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.
+
+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
+----------
+
+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 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.
+
+(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 GelRed/GelGreen fluorescence spectra. Credit Biotium Inc,
+reproduced without permission.](gelred_gelgreen){width="50%"}
+
+Simple CMOS image stacking detection
+------------------------------------
+
+### ()
+
+florescein An f1.2 lens is used.
+
+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 \[filter leakage; in their paper they refer
+to this as noise, distinct from image sensor noise\]\".
+
+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
+
+a 30-s exposure on a cheap camera also almost got there.
+
+An ELP-brand camera USB100W05MT (a common choice in industrial systems)
+with an OV9712 sensor was used. guvcview to capture.
+
+From luminescent techniques reported previously, DSLR sensors with long
+exposure times are sensitive
+
+from \[\], using ImageJ () to stack. works great on cheap cmos cameras -
+interestingly doesn't work at all on more expensive cameras. - almost
+good enough - great for diagnostics
+
+interestingly, the fact that the camera has color is quite valuable;
+filtering the green out can increase sensitivity a lot
+
+Photomultiplier photon wavelength discrimination
+------------------------------------------------
+
+Anyone familiar with photomultiplier tube use with
+
+lest you think I know what I'm talking about, I laboured under the
+assumption that the pulse height somehwo
+
+() \"The photomultiplier tube outputs an electrical charge in proportion
+to the amount of this scintllation, as a result, the output pulse height
+from the photomultiplier \"
+
+\"Does anyone know of a circuit that can discriminate color PMT\"?
+
+only works if scintillator
+
+THE pulse height is equal to the input energy! The PMT can be made
+color-sensitive; just subtract
+
+I thought the pulse height
+
+three counters? one above, one at? a lock-in amp would be better\...
+
+fast diode thresholding might work
+
+use one of the stm32f0 boards with comparators
+
+Possible artifacts and deficiencies in 
+--------------------------------------
+
+This arrangement is patently unsuitable for producing scientific
+results, as it has not even been calibrated against a DNA ladder.
+Positive control samples were generated by cracking phage capsids of a
+known titer in an autoclave and then mixing 1:1 with 1/4000 GelGreen. If
+some other process
+
+The sample passed through a microfludic channel.[^2] Adsorption
+
+Instruments should not produce a continuous stream of results.
+
+Preparation and use
+-------------------
+
+Undiluted GelGreen fluorophore (delivered at 10,000x concentration in a
+neat little screw-cap) was kept at room temperature as advised (to
+prevent crystallization or precipitation)[^3]. The fluorophore stock
+solution was prepared by diluting 2.5 microliters of GelGreen in 10 mL
+distilled water in a 15 mL screw-cap Falcon tube (McMaster-Carr
+\#7979T33) producing a weakly orange solution of \"1/4000\" dilution and
+stored at room temperature. Both were kept in light-tight metallized
+bags when not in use.
+
+solution of GG in distilled water mixed 50/50 with the sample ("1/8000")
+worked great in our case.
+
+The autosampler withdrew approximately 50 microliters of mixed solution
+from the 0.4 mL stock tube (referred to as PG1), which was then injected
+into an empty 1.5 mL empty sample tube. quantified.
+
+Cuvette
+-------
+
+The custom 1 microliter slide cuvette used for initial testing was CNC
+machined from 3 mm transparent Lexan-brand polycarbonate[^4]. After much
+mystified head-scratching, it was found that the Lexan substrate was
+very highly auto-fluorescent and completely overwhelmed the meagre DNA
+signal. This occurred with a separate coupon of Lexan, but was not
+observed with clear acrylic or polycarbonate. . That said, it has been
+reported that polycarbonate does not auto-fluoresce significantly more
+than acrylic (), so it is possible that some other effect led to this
+result.
+
+1.5 mL Eppendorf-type clear polypropylene microcentrifuge tubes
+(Carolina Premium Sterile Centrifuge tubes, 215245, believed to be MTC
+Biotech SureSeal S).
+
+Microwell plates used for luminescence are usually opaque white to
+reflect the few precious photons: fluorescence plates are typically
+opaque black.
+
+Lesson learned: beware autofluorescence
+
+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.
+
+Due to their coherent emission, eye protection or suitable interlocks
+are important when working with lasers. (Are tightly filtered LEDs
+coherent?)
+
+lasers are available.
+
+() recommends
+
+### Diode laser tests
+
+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.
+
+(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).
+
+Cree recommends staying below 300% of the continuous power when
+modulating an LED.
+
+A white LED with a high color-rendering index (DK) seemed to have more
+power in the blue passband; however, green leakage around the filter was
+too strong.
+
+Arranging the light source physically at right angles can be
+challenging. Using a plastic fiber optic assists in positioning
+\"because of the small numerical aperture\". The fiber optic ()
+
+Even a blue LED is visibly green through a
+
+Arranging the light source and detector optical paths at right angles is
+the first line of defense against excitation light bleed-through. In
+testing, is echoed by ()
+
+> Finally, it is possible to excite a sample from one side and collect
+> the fluorescence from the opposite side (Fig. 2.4.3D). While some
+> instruments, such as microplate readers, use this approach, it is rare
+> because it is difficult to completely prevent scattered excitation
+> light from reaching the detector.
+>
+> Even when thin-film filters with extremely high blocking are used,
+> high-angle scattered light can make it through the emission filter,
+> since, as shown below, the spectrum of the filter shifts to shorter
+> wavelengths for light at higher angles of incidence, thus causing the
+> shifted emitter band to overlap with the excitation wavelength band.
+
+() \"These filters should also be specified to have very low ripple in
+the passband, since the narrow laser lines of some lasers (especially
+semiconductor diode lasers) can drift over time or with changing
+environmental conditions, thus resulting in fluctuations of the filtered
+laser power.\"
+
+Filters
+-------
+
+Certainly the most critical aspect of any fluorescence technique is the
+filter set used.
+
+Important characteristics include transmission % inside the passband,
+optical density outside the passband, and sharp edges without long tails
+crossing the so-called Stokes shift() between absorption and emission.
+Some filters have ripple far from the edge of interest which must be
+taken into account when assessing the overall filtering performance.
+
+()
+
+() contributes additional information
+
+Half-inch diameter laser line filters were used here for reasons of
+cost. As of this writing, a set of quality GFP filters costs about 4
+times as much as a laser line set. Due to narrower pass-bands, perhaps
+1/8 optical performance can be expected; in a photon-counting mode,
+however, there appears to be ample sensitivity remaining.
+
+Superior filters can almost certainly be found; these were chosen for
+convenience.
+
+Gel-docs , with spectra specified using the Wratten scale. These filters
+alone do not seem to have optical density values sufficient for
+amplification-free quantitation at these levels.
+
+Most commercial microscopes use dichroic beamsplitters, allowing the
+excitation and emission beam to go through the same objective, a
+technique known as epifluorescence (epi- means same-side). The dichroic
+only removes the excitation to the 1% level - you still need the
+dielectric filters, and they're really expensive. With fiber optic
+excitation at right angles to the objective, excitation scattering was
+low enough that the dichroic was unnecessary.
+
+The microscope itself was largely incidental, providing only a base. The
+small focal length and aperture of the
+
+A 10/0.25 (10x magnification, 0.25 numerical aperture) objective was
+used.
+
+Units reported as AU
+
+### Excitation filter used
+
+One filter, an
+
+(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.
+
+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:
+
+Note the confusion that can occur when specifying passband width as
+$\pm$ versus FWHM.
+
+This is a long-pass edgepass filter. This is known as a Wratten or Gel
+filter; the \#16 is known as the Wratten number. () has a table of
+Wratten filter spectra; the cut-on wavelength (wavelength of 50%
+transmission) is 530 nm for the \#15 and 540 nm for the \#16 ($\pm$
+circa 3 nm), with 90% maximum transmission. One layer of Kapton tape was
+wrapped around the filter to protect other optics from the sharp edges.
+
+Tiffen-brand filters consist of two glass panes sandwiching a plastic
+membrane that contains the dye proper. They can be cut to size
+
+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.
+
+in series with
+
+In general, gel filters appear to have a softer taper
+
+ThorLabs has series of colored glass (\"schott glass\") filters
+
+### 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.
+
+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 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
+-----------
+
+![image](fluorescence/PMT){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
+
+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]
+
+() ()
+
+take note of potentiometer positions
+
+sketches of light source in inkscape
+
+Atmospheric pressure glow discharge (APGD) plasma generation and surface
+modification of aluminum and silicon si (100)
+
+huh! basic Argon discharge emits sharply at 488 nm - with a good fiter,
+might work out!
+
+man, most white LEDs just have a notch taken out at 488! That's so
+strange!
+
+The problem with lasers is that the only ones with power in the right
+spectrum are from china or really expensive.
+
+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.
+
+In fact, if we use a bunch of LEDs, this'll help to
+
+the CRI of the LED determines the amount of cyan light
+
+Clearance section at Edmund Optics is pretty great
+
+dichroic not needed - only does a first order-of-magnitude cut, mainly
+important
+
+custom acrylic lightpipe?
+
+Time-domain or time-correlated photon counting
+----------------------------------------------
+
+An even better Phase-shift time domain fluorimetry. Iwata use a 20 MHz
+DSO to measure a 5 ns $\tau$ fluorophore. However, this involves a light
+source with a fast modulation bandwidth; and in the implementation they
+describe, the PMT must be in the voltage mode.
+
+Called time-correlated photon counting.
+
+Another neat technique is to add I/Q
+
+Then your
+
+While there are ways to deconvolve a slow falling edge, there's another
+problem: per time interval, the time spent in the excitation-off,
+flurophore exponential decay time is proportional to the frequency of
+the excitation light. If you're only getting 1000 photons from the
+sample per second, the dye lifetime is 5 ns, and you're turning the
+light on and off at 1 MHz, you're only getting a \[\] photons from the
+exponential decay region. Even with a long exposure, that doesn't seem
+to be enough to pick up the decay.
+
+It is also possible to perform flashbulb. () abuse of pmts. Note that as
+long as average current limits are obeyed, PMTs are happy to endure very
+high pulse currents, like flashbulbs for exciting; no shutters or
+anything required. Gating the photomultiplier HV is another technique.
+Figure out the average current limit for your PMT, the expected voltage
+based on your load resistor value and watch that it never exceeds it.
+
+Another way to pick up this phase shift is to make your lock-in
+amplifier phase-sensitive - very simple (a 2x clock put into a flip-flop
+divider, then the middle). This also wasn't nearly good enough,
+completely useless.
+
+quadrature input
+
+gel tampering
+
+()
+
+One might expect, given that white noise has an expectation value of
+zero, that this technique would average out to 0, and the signal would
+be linearly proportional to time. However, a 1-dimensional random walk
+with a uniform step size of 1 is expected to end up $\sqrt{N}$ units
+away from the origin after N steps. In the same time, a fluorophore
+emitting at a rate of $r=1$ counts per step has an expected value of N
+counts (a Poisson distribution has a mean equal to its parameter).
+Therefore, $\text{SNR} = \frac{N}{\sqrt{N}} = \sqrt{N}$. Longer exposure
+times would therefore provide better precision, but with diminishing
+returns.
+
+A more comprehensive consideration ()
+
+However,
+
+it has been noted that water is a good fluorescence quencher and that
+this might be how
+
+If time-domain filtering is sufficient,
+
+Low background is required.
+
+Contrary to standard microscopy, you want as little excitation light to
+enter the objective as possible. Using the existing
+
+the input edge is considered a clock; the minimum pulse width limits
+apply, but are not clearly specified in the datasheet. In this case
+(1/155 mhz) = 6 ns.
+
+However, we had no luck with precipitation.
+
+With the setup we're using and the small quantities, the excitation
+light is somewhere around  $10^5$ times as powerful as the emission.
+This doesn't seem to be a big issue gel-docs, picking out bands on gels
+- they don't usually seem to use excitation filters; however, to get the
+excitation bleed-through low enough to do this quantitative assay, the
+bleed-through must be really low, and in our testing proper dielectric
+filters are required on both the excitation and emission sides.
+
+There are a few sources of noise:
+
+PMT dark counts Some be filtered out by judicious use of comparator
+pulse height threshold, the lock-in takes care of it. A non-issue in our
+case. Ambient light leakage It'll be fine. Even with the room lights
+Using microscope optics and the lock-in, essentially a non-issue for
+use, surprisingly. Some fluorescence microscopes use micron-size
+apertures to limit the depth of field to avoid Putting
+
+make Dia diagram of system
+
+The PMT was at maximum sensitivity (in fact, slightly above max voltage)
+
+You might be wondering why the filters are even required - why not just
+subtract the excitation bleed-through with a control? Unfortunately, any
+miniscule variation in the scattering of the excitation will be orders
+of magnitude larger than the fluorescence signal you're looking for.
+
+Some papers discuss adding a third chopper or gate period or
+photomultiplier or to measure the drift of the excitation light source.
+Putting some feedback in the loop would probably
+
+Silicon photomultipliers like ON's C-Series are almost certainly
+sufficient for this application, eliminating the HV requirements of PMTs
+at the cost of a smaller active area, requiring a larger lens to collect
+
+half-life is 0.693 times the average lifetime.
+
+Simple spectrally-filtered intensity is good enough.
+
+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
+
+Literature review
+-----------------
+
+### ()
+
+[^1]: pulse\_1.pnw line 2567
+
+[^2]: pulse\_1.pnw lines 2517
+
+[^3]: pulse\_1.pnw line 1367
+
+[^4]: pulse\_1.pnw line 1719, 1755
+
+[^5]: pulse\_1.pnw line 1879
+
+[^6]: pulse\_1.pnw line 1879
diff --git a/generate.sh b/generate.sh
index 1852e9a..e284997 100644
--- a/generate.sh
+++ b/generate.sh
@@ -1,2 +1,3 @@
 #pandoc ../../documents/fluroescence.md -o fluorescence.html
-pandoc --filter pandoc-citeproc --bibliography=../../documents/references.bib -s ../../documents/fluorescence.tex -o paper.md
\ No newline at end of file
+pandoc --filter pandoc-citeproc --bibliography=../../documents/references.bib -s ../../documents/fluorescence.tex -o README.md
+pandoc --filter pandoc-citeproc --bibliography=../../documents/references.bib -s ../../documents/fluorescence.tex -o paper.pdf
\ No newline at end of file