Combining optical trapping in a microfluidic channel with simultaneous micro-Raman spectroscopy and motion detection

Since their invention by Ashkin optical tweezers have demonstrated their ability and versatility as a non-invasive tool for micromanipulation. One of the most useful additions to the basic optical tweezers system is micro-Raman spectroscopy, which permits highly sensitive analysis of single cells or particles. We report on the development of a dual laser system combining two spatial light modulators to holographically manipulate multiple traps (at 1064nm) whilst undertaking Raman spectroscopy using a 532nm laser. We can thus simultaneously trap multiple particles and record their Raman spectra, without perturbing the trapping system. The dual beam system is built around micro-fluidic channels where crystallisation of calcium carbonate occurs on polymethylmethacrylate (PMMA) beads. The setup is designed to simulate at a microscopic level the reactions that occur on items in a dishwasher, where permanent filming of calcium carbonate on drinking glasses is a problem. Our system allows us to monitor crystal growth on trapped particles in which the Raman spectrum and changes in movement of the bead are recorded. Due to the expected low level of crystallisation on the bead surfaces this allows us to obtain results quickly and with high sensitivity. The long term goal is to study the development of filming on samples in-situ with the microfluidic system acting as a model dishwasher

Multi-plane remote refocussing epifluorescence microscopy to image dynamic Ca2+ events

Rapid imaging of multiple focal planes without sample movement may be achieved through remote refocusing, where imaging is carried out in a plane conjugate to the sample plane. The technique is ideally suited to studying the endothelial and smooth muscle cell layers of blood vessels. These are intrinsically linked through rapid communication and must be separately imaged at a sufficiently high frame rate in order to understand this biologically crucial interaction. We have designed and implemented an epifluoresence-based remote refocussing imaging system that can image each layer at up to 20fps using different dyes and excitation light for each layer, without the requirement for optically sectioning microscopy. A novel triggering system is used to activate the appropriate laser and image acquisition at each plane of interest. Using this method, we are able to achieve axial plane separations down to 15 ????m, with a mean lateral stability of ≤ 0.32 ????m displacement using a 60x, 1.4NA imaging objective and a 60x, 0.7NA reimaging objective. The system allows us to image and quantify endothelial cell activity and smooth muscle cell activity at a high framerate with excellent lateral and good axial resolution without requiring complex beam scanning confocal microscopes, delivering a cost effective solution for imaging two planes rapidly. We have successfully imaged and analysed Ca2+ activity of the endothelial cell layer independently of the smooth muscle layer for several minutes

Towards a high-throughput real-time confocal microfluidic system for monitoring absorbance spectra in mixed-phase chemical reactions

In this work, we present a compact, real-time absorbance spectroscopy instrument, designed with a particular focus on taking spectroscopic readings in a microfluidic channel environment and selectively analysing an active volume in this channel by adding confocality. There are time-saving advantages to industry in carrying out and monitoring chemical reactions in a high-throughput microfluidic environment as opposed to manually mixing and then analysing chemicals. In this paper, we use absorbance spectroscopy to investigate a particular complex mixed-phase reaction (specifically, the reaction of colloidal diluted hair dye with an oxidising agent and catalyst), which has an additional complication of oxygen gas being released then trapped in the mixture as the reaction progresses. We find that the results obtained using our instrument are comparable to those obtained in a standard spectrometer. Oxygen bubbles formed in the reaction, however, present a significant obstacle to obtaining the correct sample depth in narrow flow channels. This is mitigated through the use of a camera which views the reaction between the glass and liquid through the use of remote reimaging, allowing the bubbles to be detected via histogram analysis while spectroscopy takes place enabling rogue readings to be removed, and also serves to monitor the overall density of the ‘gassy colloidal’ mixture produced