Coherent Raman microscopy (CRM) is a powerful nonlinear optical imaging technique based on contrast via Raman active molecular vibrations. CRM has been used in domains ranging from biology to medicine to geology in order to provide quick, sensitive, chemical-specific, and label-free 3D sectioning of samples. The Raman contrast is usually obtained by combining two ultrashort pulse input beams, known as Pump and Stokes, whose frequency difference is adjusted to the Raman vibrational frequency of interest. CRM can be used in conjunction with other imaging modalities such as second harmonic generation, fluorescence, and third harmonic generation microscopy, resulting in a multimodal imaging technique that can capture a massive amount of data. Two fundamental elements are crucial in CRM. First, a laser source which is broadband, stable, rapidly tunable, and low in noise. Second, a strategy for image analysis that can handle denoising and material classification issues in the relatively large datasets obtained by CRM techniques. Stimulated Raman Scattering (SRS) microscopy is a subset of CRM techniques, and this thesis is devoted entirely to it.
Although Raman imaging based on a single vibrational resonance can be useful, non-resonant background signals and overlapping bands in SRS can impair contrast and chemical specificity. Tuning over the Raman spectrum is therefore crucial for target identification, which necessitates the use of a broadband and easily tunable laser source. Although supercontinuum generation in a nonlinear fibre could provide extended tunability, it is typically not viable for some CRM techniques, specifically in SRS microscopy. Signal acquisition schemes in SRS microscopy are focused primarily on detecting a tiny modulation transfer between the Pump and Stokes input laser beams. As a result, very low noise source is required. The primary and most important component in hyperspectral SRS microscopy is a low-noise broadband laser source.
The second problem in SRS microscopy is poor signal-to-noise (SNR) ratios in some situations, which can be caused by low target-molecule concentrations in the sample and/or scattering losses in deep-tissue imaging, as examples. Furthermore, in some SRS imaging applications (e.g., in vivo), fast imaging, low input laser power or short integration time is required to prevent sample photodamage, typically resulting in low contrast (low SNR) images. Low SNR images also typically suffer from poorly resolved spectral features. Various de-noising techniques have been used to date in image improvement. However, to enable averaging, these often require either previous knowledge of the noise source or numerous images of the same field of view (under better observing conditions), which may result in the image having lower spatial-spectral resolution. Sample segmentation or converting a 2D hyperspectral image to a chemical concentration map, is also a critical issue in SRS microscopy. Raman vibrational bands in heterogeneous samples are likely to overlap, necessitating the use of chemometrics to separate and segment them.
We will address the aforementioned issues in SRS microscopy in this thesis. To begin, we demonstrate that a supercontinuum light source based on all normal dispersion (ANDi) fibres generates a stable broadband output with very low incremental source noise. The ANDi fibre output's noise power spectral density was evaluated, and its applicability in hyperspectral SRS microscopy applications was shown. This demonstrates the potential of ANDi fibre sources for broadband SRS imaging as well as their ease of implementation. Second, we demonstrate a deep learning neural net model and unsupervised machine-learning algorithm for rapid and automated de-noising and segmentation of SRS images based on a ten-layer convolutional autoencoder: UHRED (Unsupervised Hyperspectral Resolution Enhancement and De-noising). UHRED is trained in an unsupervised manner using only a single (“one-shot”) hyperspectral image, with no requirements for training on high quality (ground truth) labelled data sets or images.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/42677 |
| Date | 16 September 2021 |
| Creators | Abdolghader, Pedram |
| Contributors | Stolow, Albert |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Attribution-NonCommercial-ShareAlike 4.0 International, http://creativecommons.org/licenses/by-nc-sa/4.0/ |
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