Application note - Detection of micro particles using OptoReader

Optoreader

Set-up: OB1 flow controller + OptoReader

The detection of micro particles in a flow is a routine task in many domains, e.g. cell biology, chromatography, medical and environmental research, etc. This task is commonly performed on conventional epi-fluorescence microscopy which is highly precise but is space consuming, require important investment and is confusing for non-expert.

OptoReader offers the highest fluorescence sensitivity and real-time processing capacity within a compact design based. The use of optical fiber to both illuminate and collect light allows OptoReader to function in tiniest space.

The main challenge in the detection of micro scale object using optical fiber is the stringent light coupling condition with small core fiber as the latter is required to produce micro scale detection region.  The option of laser source allows the OptoReader to produce light spot in micro scale while maintaining very high brightness.

In this application note, we show case the detection of 2 µm diameter fluorescent beads  using the Optoreader equiped with laser source. 

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Instruments & Components List

The experiments shown in this application note have been realized only with Elveflow’s instruments and accessories. The microfluidic channel is made by soft lithography on PDMS and supported by glass substrate. The width and height of the channel is 50 µm and 60 µm, respectively. For any advice on your research project and experimental needs, do not hesitate to consult our team of specialists.

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Sample reservoirs

Our complete line of standard and specifically designed microfluidic reservoirs provides a number of solutions that will meet any of your requirements.

Experimental set-up

2µm diameter fluorescent latex beads are obtained from Sigma-Aldrich (L4530). The initial commercial solution is diluted to obtain a solid content of about 0.025%. This solution is run through the microfluidic where the fluorescence detection is performed. The liquid flow is controlled using the Elveflow® OB1 Mk3® flow controller.

OptoReader with laser source is used to measure the fluorescence signal from the 2 µm beads. A light spot of 10 µm diameter and a numerical aperture of 0.68 is projected onto the microfluidic channel and consists the detection region. This is archived by using an optical fiber of core diameter 50 µm and a focalization optics with 5 fold magnification mounted at the free end of the fiber. The output light intensity is 1.5 mW. Given the solution concentration and the illumination geometry, there are in average 0.16 particles within the illuminated volume.

The below diagram shows the microfluidic setup used for this application note.

Fluorescence particle detection with OptoReader and Ob1

Fluorescence particle detection with OptoReader and Ob1 Mk3

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Both the OB1 Mk3 and the OptoReader are controlled by the Elveflow® Smart Interface. This software offers integrated control for the whole range of the Elveflow products with optional modules for cross-communications between the devices. The acquisition rate of OptoReader is set to 100 kHz.

Results

Fluorescence particle signal on OptoReader

Fluorescence particle signal detected with OptoReader.

The fluorescent beads crossing the detection region give rise to peaks in the detected signal as shown in the left figure. These peaks do not have the same height indicating the aggregates of different number of particles are detected.

When input pressure is set to 350 mbar, we observe an event rate of 3 kHz as shown in the red curve. At 2000 mbar (maximum pressure sustained by the used microfluidic chip), an event rate of 15 kHz is observed as shown in the blue curve.

We can infer the flow rate of the carrier fluid assuming that the beads are carried at the same speed as the liquid. Knowing the particle concentration and the event rate, the flow speed can be determined by the following formula:

where v is the flow speed, f is the event rate, d is the diameter of a bead and s is the solid content. Hence, the flow rate is about 0.44 m/s (80 µl/min) and 2.21 m/s (400 µl/min) in the cases of f = 3 kHz and 15 kHz, respectively.

Furthermore, by varying the applied pressure, one can establish the relation between the flow speed and the applied pressure. The left figure below shows the variation of the event rate with respect to the applied pressure:

Particle count vs applied pressure OptoReader

Dependence of the event rate with respect to the applied pressure

Particle size statistics with OptoReader

Width of the peaks measured when the event rate is 3kHz (red curve)

Knowing the flow rate, it is possible to perform statistical study about the size of the signal peak associated with the fluorescence. A histogram of the width of the peaks is given in the right figure. The peak width with the most highest occurrence is between 30 µm and 40 µm. This size is close to and could reflect the average width of the illuminated volume as shown in the experimental set-up diagram above.

Conclusion

OptoReader is capable of very sensitive fluorescence detection. The option of laser source allows to probe micro scale region while maintaining bright illumination intensity. We show case here the detection of micro scale fluorescent latex beads upto the rate of 15 kHz. The detection rate of these beads can be used estimate the speed of the flow. The measured width of the peaks is in good agreement with the average width of the illuminated volume.

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