New Mexico State University: Time-Resolved 해시 게임 바카라 Cytometry

The Challenge

Professor Jessica Houston’s research group at New Mexico State University investigates the unique ways in which 해시 게임 바카라 measurements can be used in해시 게임 바카라 cytometry.An important area of focus for the group is metabolic mapping of cancer cells – assessing their vitality – in the presence of chemotherapeutics, using fluorescent life해시 게임 바카라 measurements. Another key application targets fundamental cell signaling and receptor activity, using Förster (fluorescence) resonance energy transfer (FRET) and fluorescence proteins.

Houston notes that time-resolved 해시 게임 바카라 cytometry is distinctly different to most existing 해시 게임 바카라 cytometry applications which rely on steady-state fluorescence intensity data to perform counting and sorting functions. And the commercially available 해시 게임 바카라 cytometers optimized for steady-state data are not suitable for her research. So as a first step, her group had to build their own 해시 게임 바카라 cytometer.

There are two well-known ways to measure fluorescence lifetimes: in the time domain using a pulsed laser or other light source, or in the frequency domain. Houston explains, “We need a high throughput 해시 게임 바카라 cytometer in order to non-destructively count and/or sort huge populations of cells, e.g., for studying metastatic cancer cells in the blood for example. And time-domain measurements using photon counting would not support the necessary throughput. So we decided to work in the frequency domain, where the established method is to apply a modulation to the excitation laser and correlate the observed phase shift in the modulated fluorescence emission.”

The Solution

The Houston group wanted the ability to use a lot of different fluorophores in their work in addition to looking at endogenous fluorescence from 해시 게임 바카라tabolites like NADH and FAD. So they needed multiple laser wavelengths to efficiently excite all these different targets. They also needed lasers with good mode quality, i.e., low M2, as well as high-stability output, in terms of both beam pointing and power noise.

After careful evaluation, they decided to use OBIS lasers from Coherent. Houston notes, “We currently have several OBIS lasers at wavelengths including 375 nm, 405 nm, 488 nm, and 633 nm. These smart lasers are perfect for our work. In addition to their high-quality output, they support the fast direct digital modulation our work needs, plus it is so easy to add and remove these plug & play lasers which all have identical form and fit.” She notes that the availability of 10s and even 100s of milliwatts is another important OBIS feature. This is because so many of her experiments are based on frequency-domain lifetime measurements, where digital modulation essentially drops the laser power at the 해시 게임 바카라 cell by 50%.

The Result

Houston states that her custom-built 해시 게임 바카라 cytometer and her growing collection of OBIS lasers have proved to be a successful combination and have already delivered plenty of data for both her metabolic profiling studies as well as the FRET work.

For metabolic profiling, she cites the example of NADH where the measured fluorescence life해시 게임 바카라 is dependent on whether this metabolite is primarily in a bound or unbound state in the cell being assessed. This correlates with how the cell is generating ATP, the energetic molecule powering the majority of cellular activity. Metabolic activities in normal cells rely primarily on mitochondrial oxidative phosphorylation (OXPHOS) to generate ATP. But in many types of cancerous cells, glycolysis plays a larger role in ATP production, and OXPHOS capacity is reduced. So the Houston group can use the NADH life해시 게임 바카라 as a quantitative metric for how cells are responding to novel treatments for example.

The group are also using the system for fast acquisition of quantitative FRET data. They are analyzing this data to better understand protein-protein interactions, for protein conformational studies, and to probe enzy해시 게임 바카라 actions. Houston notes, “We can look at things like whether a donor fluorophore is in a quenched state of not. We can also capture both donor emissions and receptor emission. Again, this data can indicate the effectiveness of cancer therapeutics. Bottom line, the OBIS lasers and our high-throughput cyto해시 게임 바카라ter really are providing a wealth of data.”

References:

1. J. Sambrano, F. Rodriguez, J. Martin, and J. P. Houston, "Toward the Development of an On-Chip Acoustic Focusing Fluorescence Lifetime 해시 게임 바카라 Cytometer," Frontiers in Physics, 2021 9:253https://www.frontiersin.org/article/10.3389/fphy.2021.647985
2. A. Bitton, J. Sambrano, S. Valentino, and J. P. Houston, "A Review of New High-Throughput Methods Designed for Fluorescence Life해시 게임 바카라 Sensing From Cells and Tissues," Frontiers in Physics, 2021 9:163https://www.frontiersin.org/article/10.3389/fphy.2021.648553
3. K. Nichani, J. Li, M. Suzuki, and J. P. Houston, “Evaluation of caspase-3 activity during apoptosis with fluorescence life해시 게임 바카라-based cytometry measurements and phasor analyses,” Cytometry A, 2020 97(12):1265-1275;PMC7738394  
4. F. Alturkistany, K. Nichani, K. D. Houston, and J. P. Houston, “Fluorescence lifetime shifts of NAD(P)H during apoptosis measured by time-resolved 해시 게임 바카라 cytometry,” Cytometry A  2019; 95(1):70-79PMC6587805 

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