UV Raman Microspectroscopy: Spectral and Spatial Selectivity with Sensitivity and Simplicity

Abstract

The high sensitivity, selectivity, spatial resolution, and ease of operation of UV Raman microspectr oscopy is demonstrated with the use of a new highly efficient UV Raman microspectrometer with excitation at 244 nm. Single spectrograph dispersion combined with special new filters for the rejection of Rayleigh scattering improves the throughput efficiency by a factor of approximately 4 in comparison to a triple-stage spectrograph. The instrument has a spatial resolution of approximately 3 μm × 9 μm in the lateral (X–Y) plane, and 10 μm or less in the axial (Z) plane. UV resonance Raman spectra of nucleic acids are selectively excited from spatially resolved areas of a single paramecium by using low continuous-wave (cw) excitation powers and short accumulation times to minimize sample damage. High signal-to-noise Raman spectra are excited from spatially resolved areas of chemical-vapor-deposited (CVD) diamond films. We demonstrate, for the first time, the ability to probe the spatial distribution of the nondiamond carbon impurities in CVD diamond films. The amorphous carbon band at ∼ 1553 cm−1 is resolved from the normally broad ∼ 1600-cm−1 nondiamond carbon band.

Deep ultraviolet resonant Raman imaging of a cell

“…33 There are some studies reporting DUV Raman microspectroscopy of cells, but to get sufficient S/N, the area for spectral acquisition was relatively large. 30,[34][35][36] One report showed 3 × 9 μm 2 pixel-based microspectroscopy of paramecium cells, 35 while another study on plant samples used 5 μm 2 pixels. 36 Sub-micron resolution is essential for molecular imaging of a cell.…”

Deep‐Ultraviolet Biomolecular Imaging and Analysis

“…CCD camera became used in the 1990s . Filters also became available for rejecting Rayleigh scattering at that time . However, these optical devices were not as good as present ones; lasers were large and unstable, detectors did not have large QEs, and optical elements had low transmittance or reflectance.…”

The power distribution advantage of fiber‐optic coupled ultraviolet resonance Raman spectroscopy for bioanalytical and biomedical applications

“…However, the increase in the illuminated area by a factor of four allows an increase in the total power at the sample by a factor of 16, providing an overall advantage of almost an order of magnitude in total signal collected, while maintaining the same power density and therefore the same level of safety as the 150 µm fiber. The comparison of a probe using a 600 µm excitation fiber routinely employed in FO-UVRRS with a 25 µm spot, as is found in UV Raman microscopy, 15,28 results in an allowable increase in power by a factor of 576 while the collection efficiency drops by only a factor of 3.25 for the same analyte concentration. The result is that an advantage of over two orders of magnitude in total signal collected can be realized by using the larger diameter fibers provided the laser power is available to maintain the same power density.…”

“…100-200 µm are required. 28,29 If the power in the excitation beam exceeds a few milliwatts (well below the output power of currently available UV lasers), the resulting focused power density at the sample can easily exceed the damage threshold for many biomolecules. Thus, the total signal obtainable with conventional UVRR configurations is limited by a critical tradeoff between the need to focus the incident radiation to obtain an adequate signal-to-noise ratio (SNR) and the need to minimize photo-induced damage to the sample.…”