Is photobleaching necessary for Raman imaging of bone tissue using a green laser?

Abstract

Raman microspectroscopy is widely used for musculoskeletal tissues studies. But the fluorescence background obscures prominent Raman bands of mineral and matrix components of bone tissue. A 532-nm laser irradiation has been used efficiently to remove the fluorescence background from Raman spectra of cortical bone. Photochemical bleaching reduces over 80% of the fluorescence background after 2 h and is found to be nondestructive within 40 min. The use of electron multiplying couple charge detector (EMCCD) enables to acquire Raman spectra of bone tissues within 1-5 s range and to obtain Raman images less than in 10 min.

Fast Line-Based Imaging of Small Sample Features

“…organization of bone at the molecular level has been successfully explored using Raman microspectroscopy and Raman imaging [1,3,4,5]. These studies, as well as others in the literature, have begun to shed light on the molecular mechanisms of bone failure and response under both normal and diseased states.…”

“…Hence, our set of interesting pixels is P = {(1, 1), (1,2), (1,3), (3,3), (4, 3)}. Our task is to find the minimum number of columns and/or rows to scan in order to image all 5 black pixels.…”

Estimating the number of pure chemical components in a mixture by maximum likelihood

“…The circular beam profile of the Millenia II laser is reshaped into a line using a Powell lens and focused through a 4x/0.20 NA infinitycorrected objective (Nikon). For visible Raman hyperspectral imaging, a single axis scanning mirror (64240H, Cambridge Technology, Inc., Cambridge, MA) was used [28]. A LabVIEW (National Instruments, Austin, TX) program controlled the mirror's position by adjusting the voltage sent to the mirror control board through a 12-bit digital-analog converter.…”

“…Raman spectra were collected using two different systems: a Raman microprobe optimized for collection in the nearinfrared (NIR) [13] and a purpose-built, visible Raman microscope [28]. Briefly, the NIR system consists of an epi-illumination microscope frame (Olympus, BH-2) and a 400 mW 785 nm laser (Invictus, Kaiser Optical Systems, Inc.).…”

“…Visible hyperspectral imaging of mouse bone specimens embedded in PMMA were performed using the scanning mirror described previously [28], measured over the range 800-1500 cm −1 (512 spectral values). Spectra were acquired with an integration time of 4 seconds per line; the excitation power was 300 mW.…”