Deep ultraviolet mapping of intracellular protein and nucleic acid in femtograms per pixel

Abstract

By using imaging spectrophotometry with paired images in the 200- to 280-nm wavelength range, we have directly mapped intracellular nucleic acid and protein distributions across a population of Chinese hamster ovary (CHO-K1) cells. A broadband 100× objective with a numerical aperture of 1.2NA (glycerin immersion) and a novel laser-induced-plasma point source generated high-contrast images with short (~100 ms) exposures and a lateral resolution nearing 200 nm that easily resolves internal organelles. In a population of 420 CHO-K1 cells and 477 nuclei, we found a G1 whole-cell nucleic acid peak at 26.6 pg, a nuclear-isolated total nucleic acid peak at 11.4 pg, and, as inferred by RNase treatment, a G1 total DNA mass of 7.4 pg. At the G1 peak we found a whole-cell protein mass of 95.6 pg, and a nuclear-isolated protein mass of 39.3 pg. An algorithm for protein quantification that senses peptide-bond (220-nm) absorbance was found to have a higher signal-to-noise ratio and to provide more reliable nucleic acid and protein determinations when compared to more classical 280-nm/260-nm algorithms when used for intracellular mass mapping. Using simultaneous imaging with common nuclear stains (Hoechst 33342, Syto-14, and Sytox Orange), we have compared staining patterns to deep-UV images of condensed chromatin and have confirmed bias of these common nuclear stains related to nuclear packaging. The approach allows absolute mass measurements with no special sample preparation or staining. It can be used in conjunction with normal fluorescence microscopy and with relatively modest modification of the microscope.

Deep ultraviolet resonant Raman imaging of a cell

“…The image is not very clear, but does show a characteristic distribution of RNA in a cell, which is relatively low in the nucleus and higher in the cytoplasm. 31 The results show how DUV Raman microscopy can be used for distinguishing distributions, such as DNA and RNA in a cell without any molecular labeling.…”

“…DUVoptics and light sources have been specifically developed and revamped in the past decade. Because of this, DUV studies for microscopy 31,37 lithography, sterilization, and other fields are growing. Recently, plasmonics, with the features of electric field enhancement and spatial confinement, is also budding in DUV and preliminary applications for enhanced Raman scattering were reported.…”

Intracellular protein and nucleic acid measured in eight cell types using deep‐ultraviolet mass mapping

“…However, the process of isolating nuclei, and more generally of preparing cells for flow cytometry, hinders cell-resolved measurements of other parameters such as cell morphology and intracellular protein distribution. Therefore, while fluorescence methods will certainly continue to have a dominant role in the exploration of biological heterogeneity, deep ultraviolet (UV) mass mapping (7,8) has the potential to provide cell-resolved measurement of nucleic acid and protein mass, to preserve cell morphology, and to avoid utilizing fluorescence channels that can otherwise be dedicated to tracking specific proteins of interest.…”

Deep-UV biological imaging by lanthanide ion molecular protection

“…DUV light can destroy or denature biological molecules due to absorption [10][11][12][13][14][15][16][17][18][19][20][21], which restricts its use in quantitative, repetitive, and/or high signal-to-noise ratio (SNR) analyses of target molecules in a limited detection volume by DUV imaging, especially for samples involving trace amounts. Although a variety of advanced DUV imaging techniques measuring absorption [22][23][24], resonance Raman scattering [25], fluorescence [26][27][28][29][30], and photoacoustic signals [31,32] of DUV-absorptive molecules have been developed recently, the destructive nature of DUV light limits some unique benefits of these techniques in exploiting biological molecules, which could be measured with high sensitivity if the photodegradation could be avoided. Suppressing the molecular photodegradation is essential for unlocking these limitations in the practical use of DUV light for biological imaging.…”