Flow cytometry is a widely used tool for high-throughput quantitative analysis of cell populations and intracellular content. Signals in flow cytometry arise from electrical impedance, forward or side light scattering, and fluorescence. Scattering and electrical impedance provide granularity and size/volume information, but with no chemical specificity. Fluorescent labeling acts as the primary approach for cellular analysis in flow cytometry. Nevertheless, fluorescent tags are not applicable to all cases, especially small molecules (e.g. drugs) for which labeling may significantly perturb their properties. The current application aims to fill this gap through the development of a multichannel spectral flow cytometer using stimulate Raman scattering (SRS) signal from inherent molecular vibration. The stimulated Raman scattering overcomes the low signal level in spontaneous Raman scattering. It measures the light-matter energy transfer and is therefore free of the nonresonant background encountered in coherent anti-Stokes Raman scattering. Moreover, as a nonlinear optical process it is inherently phase matched, permitting a weakly focused collinear beam geometry that is compatible with high-speed detection of flowing objects. The planned instrumentation contains two specific aims. The first is to build a single-frequency SRS flow cytometer using a femtosecond laser source. The second is to build a multichannel SRS flow cytometer by multiplex detection of spectrally dispersed SRS signals. Based on the large signal level, we expect to reach the speed of 10,000 cells per second. Performance of the label-free spectral cytometer will be tested through quantitation of fat storage in adipocytes and of drug uptake by cancer cells.

Public Health Relevance

(provided by applicant): We propose to develop a label-free multichannel spectral flow cytometer using the stimulated Raman scattering signal from inherent molecular vibration. The label-free spectral cytometer will be applied to quantify the fat storage in adipocytes and drug uptake by cancer cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21GM104681-01
Application #
8352320
Study Section
Special Emphasis Panel (ZRR1-BT-7 (01))
Program Officer
Friedman, Fred K
Project Start
2012-08-01
Project End
2015-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
1
Fiscal Year
2012
Total Cost
$192,500
Indirect Cost
$67,500
Name
Purdue University
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907
Hu, Chun-Rui; Zhang, Delong; Slipchenko, Mikhail N et al. (2014) Label-free real-time imaging of myelination in the Xenopus laevis tadpole by in vivo stimulated Raman scattering microscopy. J Biomed Opt 19:086005
Zhang, Delong; Wang, Ping; Slipchenko, Mikhail N et al. (2014) Fast vibrational imaging of single cells and tissues by stimulated Raman scattering microscopy. Acc Chem Res 47:2282-90
Hu, Chun-Rui; Slipchenko, Mikhail N; Wang, Ping et al. (2013) Stimulated Raman scattering imaging by continuous-wave laser excitation. Opt Lett 38:1479-81
Zhang, Delong; Slipchenko, Mikhail N; Leaird, Daniel E et al. (2013) Spectrally modulated stimulated Raman scattering imaging with an angle-to-wavelength pulse shaper. Opt Express 21:13864-74
Wang, Ke; Zhang, Delong; Charan, Kriti et al. (2013) Time-lens based hyperspectral stimulated Raman scattering imaging and quantitative spectral analysis. J Biophotonics 6:815-20
Zhang, Delong; Wang, Ping; Slipchenko, Mikhail N et al. (2013) Quantitative vibrational imaging by hyperspectral stimulated Raman scattering microscopy and multivariate curve resolution analysis. Anal Chem 85:98-106