The injection mix (IM) is a key component of the injection process. It requires specific preparation, and its behavior must be well understood to guarantee successful delivery. This section covers how to select and prepare the IM, configure imaging and flow measurement settings in ARYA, and interpret flow data to confirm suitability before running injection experiments.
Injection mix composition and preparation
The IM composition depends on the specific application goals. However, consider the following general recommendations:
- Fluorescent tracers in the IM enable easy monitoring of the injection process. Tracers can be used alone in the IM, co-injected with other molecules or labelling certain molecules. When selecting a tracer, consider the following factors: molecular size, working concentration, cytotoxicity, and compatibility of the fluorescence spectrum with the available imaging system.
Filter | Excitation Filter | Emission Filter | Corresponding color in the visible spectrum |
U-FUNA | 360-370 nm | 420-460 nm | Blue |
U-F39002 | 480 nm | 535 nm | Green |
U-F39004 | 540 nm (528-552) | 605 nm (578-632) | Red |
U-F49004 | 545 nm (533-557) | 605 nm (579-640) | Red |
- When co-injecting, ensure that the tracer and other IM components have similar sizes for consistent injection behavior.
Fluorescent tracer | Concentration | Excitation wavelength | Emission wavelength | Fluorescence filter |
Lucifer Yellow | 2 mg/mL | 428 nm | 544 nm | U-F39002 |
DAPI | 0.25 mg/mL | 359 nm | 461 nm | U-FUNA |
Rhodamine dextran (20kDa) | 0.25 mg/mL | 555 nm | 580 nm | U-F49004 |
FITC dextran (20kDa) | 0.25 mg/mL | 490 nm | 520 nm | U-F39002 |
- Since FluidFM Nanosyringe apertures are in the nanometer range, it is critical to avoid aggregates in the IM that could clog the cantilever. Clogging can result from:
- Aggregation (high concentration, molecular properties, incorrect buffer).
- Surface deposition (molecular properties, cantilever residues, surface adsorption).
- Protocol issues (improper handling, expired reagents).
To prevent clogging, filter the IM through a 0.22 µm filter immediately before use. If filtration is not feasible, centrifuge the IM at 16,000 x g (relative centrifugal force) at 4°C for 22 min and use the supernatant. Both methods reduce particulates that could clog the probe.
Flow Characterization
When introducing a new IM solution or changing the concentration of a previously used IM, assessing its flow characteristics with a FluidFM Nanosyringe becomes crucial. This test evaluates IM flow properties through the delivery system without requiring a cell-based experiment.
The test measures the increase of fluorescence around the aperture of the cantilever for a given time. Fluorescence intensity is assumed to be proportional to the delivered IM volume, enabling flow quantification. This analysis is essential because it determines IM stability duration within the microchannel, flow changes across sequence repetitions, and clogging event frequency. Simulating pressure sequences as for an injection can also provide information on probe lifetime during the planned experiments.
Imaging intensity settings
When using a new IM, calibrate the imaging settings to ensure:
- Minimal photobleaching.
- No signal saturation during the flow measurement.
Fluorescence image of a cantilever filled with Lucifer Yellow at 10x, showing intensity settings. (a) no saturated pixels visible, no oversaturation when applying longer pressure pulses. (b) Oversaturated pixels during flow measurement. This sample requires adjustment of the imaging parameters.
- Sufficient signal detection for low-pressure pulses.
Use 10x magnification for optimal visualization of the delivery area. To minimize the background noise, use a separate well containing a non-fluorescent solution (e.g., PBS).
The imaging settings can be saved with the Preset tool, for future use in ARYA.
Flow measurement workflow
Configure the following parameters in Measure Flow in ARYA (Settings>Workflows>Experimental workflows> Measure Flow):
- Rectangle size (pixels): Defines the region of interest (ROI) dimensions for intensity measurement.
- Initial idle time: Duration before the intensity recording begins, allowing background stabilization.
- Sequence repetitions: Number of times that a sequence will be repeated.
- Probe serial number: Automatically populated when the probe is scanned during the Preparation Advanced step. This value is included in the Results file for probe identification.
Within Measure Flow, the sequence for the flow measurement can be defined (Edit button). Sequences can be created by:
- Single pulse
- Pulse train
- Previously saved sequences
Single pulse
A single pulse consists of a pressure pulse followed by an idle pressure phase. During the idle phase, the background intensity will reduce to the initial value.
Imaging presets can be defined for the pulse and for the idle phase.
(a) Single pulse explained in phases and showed in ARYA. (b) Parameters definition for single pulse sequences in ARYA.
Pulse train
This setup uses a series of pressure pulses of equal duration followed by identical idle durations. The user can define the pressure increment for each pulse. As for the single pulse, the imaging presets can be determined for both the pulse duration and the idle phase.
Pulse train example. In between each pulse, there is an idle time where no pressure is applied and intensity decreases before next measurement takes place.
Parameters definition for pulse train sequences in ARYA
What sequence should be used to evaluate an IM?
The goal of the flow measurement is to reproduce what happens with the FluidFM Nanosyringe while injecting, not only on a given single pulse, but in longer probe use. We would recommend using the following sequence as a standard when assessing the IM:
- Pulse train, with a starting pressure of 50 mbar and with 50 mbar pressure increments (step count 6). At the very last step, include 10 min of 100 mbar of continuous pressure.
- During pulse train, use defined fluorescent imaging presets (as in section Imaging intensity settings).
- During continuous pressure step, use imaging presets with ‘shutter closed’, so that photobleaching is reduced.
Flow Data Fitting Tool
Data from the flow measurements can be analyzed with dedicated software (C:\Program Files\Cytosurge\ARYA\ FlowDataFittingTool.exe). In the Flow Data Fitting Tool, open the saved .txt file of the experiment; the data will be automatically loaded and visualized:
Flow data for Lucifer Yellow, 2mg/ml in DPBS in non-coated FluidFM Nanopipettes, shown in the dedicated fitting tool.
The software displays the following graphs:
- Intensity profile (A), showing intensity changes over time. This profile can be used to check for any clogging events during the pulse trains. For each pulse, the system fits a line for the intensity of the initial part of the pressure pulse (when the increase of the intensity is exponential). The slope fitting can be adjusted by dragging the line to the desired location.
- A table with the slope values for each pressure pulse (B). When selecting a value in the list, the corresponding pulse in the intensity profile will be highlighted.
- A graph (C) showing the flow rate vs pressure with the values obtained in table B. Outlier points can be excluded by deselecting them in the slope values table B. This plot shows if the flow decreases or increases during a pulse train.
- A summary table showing the slope values vs repetition number (D).
- A graph with the values obtained in D (E), which shows flow behavior across the multiple repetitions (decreasing flow, clogging, stable flow, etc.).
Data Analysis
Perform at least three independent flow measurements to understand the flow characteristics of a certain IM within the FluidFM Nanosyringe.
To compare different flow measurements, certain indicators must be considered:
- Verify that no clogging occurred before starting the flow measurement.
- Minimum signal-to-noise (S/N) ratio: the signal in the first pulse should be at least twice the noise level.
- No signal saturation during flow measurement.
- During data analysis, verify that slope fitting shows a linear trend with error bars of acceptable magnitude.
If one of the indicators is not met, data quality and analysis could be biased.
The following image shows an example of Lucifer Yellow (2 mg/ml in PBS) in uncoated FluidFM Nanopipettes. Flow rate slopes from three independent runs using three different probes are plotted. The graph demonstrates consistent flow rates across all tested conditions, with minimal variation
Values for the slopes for the flow measurement of Lucifer Yellow, 2mg/ml in DPBS for three independent non-coated FluidFM Nanopipettes, obtained in the Flow Data Fitting tool are plotted. For each flow measurement, the flow was measured with a five repetitions of a pulse train, with pressure pulses increasing from 50 to 300 mbar, and an idle period of 10 min and 100 mbar of continuous pressure (standard sequence previously defined).
These summarized results can give the following information about the IM:
- Stable flow
If an IM has a stable flow and shows no signs of clogging, it is ready to use. The example image of Lucifer Yellow shows a stable flow over all repetitions. Note: Probes may still clog due to cell debris. Perform regular flushing during injection assays to maintain the flow.
Flow data for Lucifer Yellow, 2mg/ml in DPBS in non-coated FluidFM Nanopipettes, shown in the dedicated fitting tool.
- Bleaching
Photobleaching may occur during flow measurements, presenting as a linear decay in fluorescence intensity across sequence repetitions without observable clogging events.
Flow data for Lucifer Yellow, 2mg/ml in DPBS in non-coated FluidFM Nanopipettes. Slow degradation in the flow through the different sequences, due to bleaching of the dye.
Bleaching can be minimized by using the lowest feasible exposure settings for the dye and reducing exposure time through imaging presets adjustments (e.g., closing the shutter or turning off the lamp).
- Immediate clogging at start of measurements
The chosen IM is unsuitable for injection. Identifying and addressing the cause of clogging may improve IM performance.
- Gradual clogging over the measurements
If an IM shows gradual clogging over the sequences, increase pressure during injection to achieve consistent injection outcomes. Ensure that gradual changes are not due to photobleaching of the dye.
- Sudden clogging during measurements
When sudden clogging is observed during the measurement, the IM can be used for a limited number of injections. High-pressure flushes between injections may help extend probe lifetime. Plan probe usage per experiment accordingly.
Flow data for FITC-Dextran 70kDa, 0.25mg/ml in water in non-coated FluidFM Nanopipettes. First three sequences show a decrease in the flow. The flow reduced drastically for the last two sequences, indicating that the probe clogged.
More information can be found in the following link: https://documentation.cytosurge.com/bot/current/workflows/measureflow/