Laser-Induced Fluorescence Emission (L.I.F.E.) as Novel Non-Invasive Tool for In-Situ Measurements of Biomarkers in Cryospheric Habitats

The overall goal of this study is to calibrate and test a novel device that can be used for in-situ measurements of biomarkers in cryospheric habitats. The device is based on Laser-Induced Fluorescence Emission technology, referred to as LIFE. The current standard sampling methods lead to physical impacts on the specimen due to quarrying, disintegration of samples by chipping and sawing, and temperature shift by melting.

These methods often lead to falsification of in-situ conditions. Therefore, a new methodology for the in-situ detection and characterization of microbial life is crucial. We have developed a novel non-invasive, non-destructive method with a high spatial and temporal resolution.

The application of a laser-induced fluorescence emission technique is based on the fact that supraglacial environments are inhabited, among other things, by phototropic organisms. These organisms can be traced by the detection of fluorescent patterns of the porphyrin-based accessory pigments chlorophyll A and phycoerythrin which are excited by a blue and green laser respectively. The portable dual wavelength kit weighs 4.5 kilograms and is used on a tripod in combination with an external computer.

The lens tube is directed towards the specimen. Then, a green five milliwatt laser hits the sample after passing a polarizing beam splitter that redirects polarized light towards the optical axis of the spectrometer. The specimen exhibits a fluorescent light illustrated in red.

Half of the collimated light passes the polarizing beam splitter and is focused through a long-pass filter which removes the laser signals. Next, the signal hits an aperture slit which consists of two adjustable razor blades. A prism spectrally separates a fine line of light orthogonal to the slit aperture before the signal is captured on a sensor.

The procedure is repeated with the blue laser. The raw data is transferred automatically to a portable computer which is also used for the software operation. It is divided in three main sections.

The exposure adjustment is done manually. Here, the correction between exposure time and signal intensity is linear. The comment field is used for the description of a sample.

In the right section, raw images are displayed as soon as the measurement is finished. This feature is crucial for immediate data evaluation in the field. Red areas indicate overexposed pixels which can be avoided by reducing the exposure time.

The 12-bit grayscale raw images show a spatial component due to the one-dimensional aperture slit and a spectral component due to the prism in front of the CCD. In response to optical constraints, the raw images are distorted. Therefore, they need to be cropped and dewarped by applying a code that recognizes the degree of distortion.

Next, the wavelength calibration is done with the help of the 532 nanometer laser. The green light is produced by frequency doubling of 1, 064 nanometer infrared laser. Both wavelengths can be detected by the CCD and therefore the spectral position of each pixel can be calculated in dewarped images.

Then, the picture is cropped down to a given wavelength range. Gray values from each pixel in a selected pixel line are counted and summed up. A gray value can range from zero to 255.

After that, every pixel line accounts for one number. In this graph, the gray value counts from each pixel line are plotted against the spatial coordinates. This allows a quantitative spatial discrimination of chlorophyll and phycoerythrin simultaneously within the sample.

Additionally, the spectral properties of a sample can be plotted from selected pixel lines. The field setup is quick and easy. Attach the instrument on a tripod.

Attach the lens tube to the device. Attach the Arduino USB cable and the camera cable. Connect the external computer with the instrument using a USB cable.

Adjust the tripod legs in a way that the lens tube covers the specimen. Run the software, describe the sample, and start a measurement run. For the pigment calibration, prepare a dilution row series from a stock solution as displayed.

The chlorophyll A stock solution needs to be diluted with acetone and phycoerythrin dilution is performed with distilled sterile water. Fifteen milliliters of each dilution step will be needed later. Protect the pigments from light by wrapping them under aluminum foil.

Store the chlorophyll in a freezer and the phycoerythrin in a fridge until further use. Next, build a rack as shown with a difference in height of 1.5 centimeters. Add five milliliters of the highest concentrated dilution in a poly-scintillation plastic vial.

This volume equates to a 15 millimeter high water column in the vial and measure the fluorescence intensity. Then, place the vial in the middle position of the rack and add another five milliliters of the same solution. Repeat the procedure with another five milliliters which equals 45 millimeters in column height.

Repeat the procedure with all dilution steps for chlorophyll and phycoerythrin. The rack and column height play an important role for the measurement since the surface of the liquids lie in the focal point of the LIFE instrument. Collect snow and ice samples from a glacier.

Additionally, collect microbial mat samples from the glacier forefield. In this study, Midtre Lovenbreen, a glacier nearby the research facilities of Ny-Alesund in the high arctic archipelago of Svalbard was chosen. Melt snow and ice samples and vacuum filter them under GF/F filters.

Note the filtered volume. Then, measure the filters with the LIFE device on four random areas each in triplicates using the green and blue laser. Calculate the overall pigment concentration by multiplying the area density with the filtered area and filtered volume.

Normalize the pigment concentration to a volume of one liter. Put the filters in a vial with 30 milliliters of acetone and store them in the dark at four degrees centigrade overnight. Next, take a vial and place it on ice prior to sonication for two minutes at 50%power in continuous mode.

Squeeze and remove the filter from the vial. The filter is no longer needed. Attach Tygon tubing to a syringe and remove the chlorophyll extraction acetone mix from the vial.

Replace the Tygon tubing with a GF5 filter holder. Transfer the solution into a quartz cuvette. After calibrating the absorbance spectrometer for acetone, place the sample containing cuvette in the spectrometer and measure the absorbance features between 400 and 750 nanometers.

Next, remove the cuvette from the spectrometer and add 200 microliters of two molar hydrochloric acid to the sample. Then, repeat the absorbance measurement in order to measure the pheophytin content in the sample. The effect of the laser measurement on the activity of bacterial communities is not described in detail yet.

Therefore, the effect has been investigated via primary and secondary production. For bacterial production, take five aliquots of our bacterial mat. Three aliquots are used for the tritium-labeled leucine uptake and two aliquots are used as controls.

Inactivate the controls with formaldehyde. Add tritium-labeled leucine to all aliquots. Repeat that procedure with all samples.

Here, the required sample amount is shown for our experimental design. Next, expose the bacterial mat with the green and blue laser as indicated in the experimental setup. Then, inactivate all samples that were not yet treated with formaldehyde.

Transfer the sample into a cryovial and add trichloroacetic acid or TCA. Centrifuge the vial at 10, 000 g for five minutes. Add scintillation liquid and put the cryovial into a poly-scintillation vial.

Analyze the samples with a liquid scintillation counter and calculate the uptake rates. For photosynthesis, prepare five aliquots as before of which two are darkened. Add the radioactive tracer NaH14 CO3 and incubate for four hours.

After incubation, stop the reaction by wrapping the samples in aluminum foil. Measure disintegrations per minute by liquid scintillation as described before. After normalizing the data for an exposure time of one second and a sample column height of 15 millimeters, the final calibration line was calculated using a Poisson regression.

The correlation between area density and photon counts has a linear character. The slope of the curve is 81.04. That means that a photon count rate of 8, 104 in a sample exposed for one second equals an area density of 100 nanograms per centimeter squared of phycoerythrin.

The standard deviation in higher concentrated samples can be explained by a self-absorption process within the sample. The chlorophyll A calibration curve shows similar characteristics and has a linear character with a slope of 8.94. Six filtered ice and snow samples were measured with the LIFE instrument before chlorophyll extraction and subsequent absorbance spectrum measurements for chlorophyll content analysis.

The samples are ordered by their chlorophyll content acquired by the absorbance spectrometry. The LIFE data show small standard deviations which suggests that the material on the filter was distributed relatively equally. The LIFE instrument underestimates the chlorophyll content of the first three filters.

The last three filters, which show a lower chlorophyll content, are overestimated by the LIFE instrument. The reason for the data disparity can be explained by the thickness of the filter cake. In thin filter cakes, illustrated in orange, low fluorescence signals are captured due to low area densities of the pigments.

The chlorophyll A signals are illustrated in red in this raw image. All gray areas in this raw image indicates that the filter itself exhibits laser-induced signals after passing the 450 nanometer long-pass filter. The software misleadingly counted this signal as chlorophyll A fluorescence.

High pigment contents were underestimated because the laser could not induce fluorescence response in chlorophyll A molecules in deeper layers of the filter cake. The laser exposure experiment indicates no significant effect on the primary and secondary productivity of bacterial mats. Neither the exposure time from five to 60 seconds nor the laser intensity ranging from five to 50 milliwatts showed any effect on productivity results.

This implies that the higher intensities and exposure times required to produce better signals would not harm the cells. Four cryoconites were measured in-situ and they're compared with chlorophyll standard solution which was measured under laboratory conditions shown in red. The spectral fluorescence peaks in blue matched with the chlorophyll standard spectrum, thus providing the concept of in-situ measurements.

LIFE measurements have been performed on soils, bacterial mats, biofilms, and cryoconites. LIFE measurements of cryoconites with a thick sediment layer are possible if the cryoconite covered surface area exceeds 12.5 centimeter squared. This type of cryoconite blocks stray light from beneath the ice.

In thin cryoconite layers, the fluorescence signals are shrouded by ambient light. Accordingly, LIFE measurements of bare ice surfaces are not possible while all other sample types can be analyzed by the LIFE instrument. Temperature changes lead to enhanced availability of liquid water which results in higher biological activity on glacier surfaces.

As a consequence, the pigment concentration may increase. It is crucial to monitor these changes and predict possible and likely future scenarios.