diff --git a/src/main/java/com/glencoesoftware/bioformats2raw/BioTekReader.java b/src/main/java/com/glencoesoftware/bioformats2raw/BioTekReader.java
index b201a6d4..79628f58 100644
--- a/src/main/java/com/glencoesoftware/bioformats2raw/BioTekReader.java
+++ b/src/main/java/com/glencoesoftware/bioformats2raw/BioTekReader.java
@@ -70,6 +70,9 @@ public class BioTekReader extends FormatReader {
   private static final String TIFF_REGEX_Z =
     WELL_REGEX + "_.+\\[(.+)_" +
     ALPHANUM + "\\]_(\\d+)_(\\d+)_([0-9-]+)?" + SUFFIX;
+  private static final String TIFF_REGEX_ROI =
+    "([A-Z]{1,2})(\\d{1,2})ROI(\\d+)_(\\d+)_(\\d+)_(\\d+)(Z(\\d+))?_" +
+    ALPHANUM + "_(-?\\d+)" + SUFFIX;
   private static final String DATE_FORMAT = "MM/dd/yy HH:mm:ss";
 
   // -- Fields --
@@ -211,22 +214,81 @@ protected void initFile(String id) throws FormatException, IOException {
     findXPTFiles(parent);
 
     String[] files = parent.list(true);
+    for (int i=0; i<files.length; i++) {
+      files[i] = new Location(parent, files[i]).getAbsolutePath();
+    }
     Arrays.sort(files);
+
+    // is there only one well in the directory?
+    // compare the well identifiers (relative file name up to first _)
+    boolean sameWell = true;
+    int endIndex = files[0].indexOf("_");
+    if (endIndex > 0) {
+      String wellCheck = files[0].substring(0, endIndex);
+      LOGGER.debug("well check string = {}", wellCheck);
+      for (int i=0; i<files.length; i++) {
+        if (!files[i].startsWith(wellCheck)) {
+          sameWell = false;
+          break;
+        }
+      }
+    }
+    LOGGER.debug("single well in {}: {}", parent, sameWell);
+    // if only one well exists, look in other subdirectories of the parent
+    if (sameWell) {
+      Location plateDir = parent.getParentFile();
+      LOGGER.debug("plate directory = {}", plateDir);
+      String[] wellDirs = plateDir.list(true);
+      ArrayList<String> allFiles = new ArrayList<String>();
+      boolean multipleDirs = true;
+      for (String well : wellDirs) {
+        Location wellDir = new Location(plateDir, well).getAbsoluteFile();
+        LOGGER.debug("looking in well directory = {}", wellDir);
+        if (wellDir == null) {
+          multipleDirs = false;
+        }
+        else {
+          String[] f = wellDir.list(true);
+          if (f == null) {
+            multipleDirs = false;
+          }
+          else {
+            for (String file : f) {
+              LOGGER.debug("  adding well file {}", file);
+              allFiles.add(new Location(wellDir, file).getAbsolutePath());
+            }
+          }
+        }
+      }
+      if (multipleDirs) {
+        LOGGER.debug("found files = {}", allFiles);
+        files = allFiles.toArray(new String[allFiles.size()]);
+        Arrays.sort(files);
+      }
+    }
+
     Pattern regexA = Pattern.compile(TIFF_REGEX_A);
     Pattern regexB = Pattern.compile(TIFF_REGEX_B);
     Pattern regexZ = Pattern.compile(TIFF_REGEX_Z);
+    Pattern regexROI = Pattern.compile(TIFF_REGEX_ROI);
+    ArrayList<WellIndex> validWellRowCol = new ArrayList<WellIndex>();
     int maxRow = 0;
-    int minRow = Integer.MAX_VALUE;
     int maxCol = 0;
-    int minCol = Integer.MAX_VALUE;
     int maxPlateAcq = 0;
     Map<Integer, Integer> maxField = new HashMap<Integer, Integer>();
 
-    for (String f : files) {
+    String matchingPath = new Location(currentId).getAbsolutePath();
+    LOGGER.trace("matching path = {}", matchingPath);
+
+    ArrayList<String> foundWellSamples = new ArrayList<String>();
+
+    for (String absolutePath : files) {
+      String f = new Location(absolutePath).getName();
       Matcher m = regexA.matcher(f);
       int rowIndex = -1;
       int colIndex = -1;
       int fieldIndex = -1;
+      int roiIndex = -1;
       int z = 0;
       int t = 0;
       String channelName = "";
@@ -261,28 +323,68 @@ protected void initFile(String id) throws FormatException, IOException {
           t = (int) Math.max(0, Integer.parseInt(m.group(8)) - 1);
           channelName += m.group(6);
         }
+        else {
+          m = regexROI.matcher(f);
+          if (m.matches()) {
+            rowIndex = getWellRow(m.group(1));
+            colIndex = Integer.parseInt(m.group(2)) - 1;
+            roiIndex = Integer.parseInt(m.group(3)) - 1;
+
+            LOGGER.trace("absolutePath = {}, roiIndex = {}",
+              absolutePath, roiIndex);
+
+            int channelIndex = Integer.parseInt(m.group(5)) - 1;
+            fieldIndex = Integer.parseInt(m.group(6)) - 1;
+            try {
+              z = Integer.parseInt(m.group(8));
+              // can have two channels with same name
+              // one with Z stack and one without
+              channelName = "Z";
+            }
+            catch (NumberFormatException e) {
+            }
+            channelName += m.group(9);
+            // recorded T index may be negative if no timepoints
+            t = (int) Math.max(0, Integer.parseInt(m.group(10)) - 1);
+          }
+        }
       }
 
-      if (rowIndex >= 0 && colIndex >= 0 && fieldIndex >= 0) {
+      if (rowIndex >= 0 && colIndex >= 0 &&
+        (fieldIndex >= 0 || roiIndex >= 0))
+      {
+        // collapse field and ROI index into single field index
+        if (roiIndex >= 0) {
+          if (fieldIndex < 0) {
+            // only ROIs, no field
+            fieldIndex = roiIndex;
+          }
+          else {
+            // both fields and ROIs
+            String key = fieldIndex + "," + roiIndex;
+            if (!foundWellSamples.contains(key)) {
+              foundWellSamples.add(key);
+            }
+            fieldIndex = foundWellSamples.indexOf(key);
+          }
+        }
+
         BioTekWell well = lookupWell(0, rowIndex, colIndex);
         if (fieldIndex >= well.getFieldCount()) {
           well.setFieldCount(fieldIndex + 1);
         }
         int c = well.addChannelName(fieldIndex, channelName);
-        well.addFile(new PlaneIndex(fieldIndex, z, c, t),
-          new Location(parent, f).getAbsolutePath());
+        well.addFile(new PlaneIndex(fieldIndex, z, c, t), absolutePath);
 
         if (rowIndex > maxRow) {
           maxRow = rowIndex;
         }
-        if (rowIndex < minRow) {
-          minRow = rowIndex;
-        }
         if (colIndex > maxCol) {
           maxCol = colIndex;
         }
-        if (colIndex < minCol) {
-          minCol = colIndex;
+        WellIndex rowColPair = new WellIndex(rowIndex, colIndex);
+        if (!validWellRowCol.contains(rowColPair)) {
+          validWellRowCol.add(rowColPair);
         }
         Integer maxFieldIndex = maxField.get(0);
         if (maxFieldIndex == null) {
@@ -292,6 +394,7 @@ protected void initFile(String id) throws FormatException, IOException {
       }
     }
     wells.sort(null);
+    validWellRowCol.sort(null);
 
     // split brightfield channels into a separate plate acquisition
     maxField.put(1, -1);
@@ -303,6 +406,8 @@ protected void initFile(String id) throws FormatException, IOException {
       for (int f=0; f<w.getFieldCount(); f++) {
         String[] fieldFiles = w.getFiles(f);
         for (String file : fieldFiles) {
+          LOGGER.trace("found file {} for well index {}, field index {}",
+            file, well, f);
           Element root = getXMLRoot(file);
           boolean brightfield = isBrightField(root);
 
@@ -401,14 +506,26 @@ protected void initFile(String id) throws FormatException, IOException {
       setSeries(s);
 
       int wellIndex = getWellIndex(s);
-      int field = getFieldIndex(s) + 1;
+      int field = getFieldIndex(s);
       BioTekWell well = wells.get(wellIndex);
       int row = well.getRowIndex();
       int column = well.getColumnIndex();
-      store.setImageName(
-        getWellName(row, column) + " #" + field, s);
 
-      List<Channel> channels = well.getChannels(field - 1);
+      String fieldName = " #" + (field + 1);
+      if (field < foundWellSamples.size()) {
+        // unpack the field/ROI indexes and store both
+        // in the image name
+
+        String[] indexes = foundWellSamples.get(field).split(",");
+        if (indexes.length == 2) {
+          fieldName = " Field #" + (Integer.parseInt(indexes[0]) + 1) +
+            ", ROI #" + (Integer.parseInt(indexes[1]) + 1);
+        }
+      }
+
+      store.setImageName(getWellName(row, column) + fieldName, s);
+
+      List<Channel> channels = well.getChannels(field);
       for (int c=0; c<getEffectiveSizeC(); c++) {
         if (channels != null && c < channels.size()) {
           Channel channel = channels.get(c);
@@ -443,12 +560,14 @@ else if (pa == 1) {
 
     int nextImage = 0;
     int[] nextWellSample = new int[(maxRow + 1) * (maxCol + 1)];
-    int totalColumns = (maxCol - minCol) + 1;
     for (int w=0; w<wells.size(); w++) {
       BioTekWell well = wells.get(w);
-      int effectiveRow = well.getRowIndex() - minRow;
-      int effectiveColumn = well.getColumnIndex() - minCol;
-      int wellIndex = effectiveRow * totalColumns + effectiveColumn;
+      int wellIndex = validWellRowCol.indexOf(
+        new WellIndex(well.getRowIndex(), well.getColumnIndex()));
+      LOGGER.debug(
+        "well #{}, row = {}, col = {}, index = {}",
+        w, well.getRowIndex(), well.getColumnIndex(), wellIndex);
+
       well.fillMetadataStore(store, 0, well.getPlateAcquisition(), wellIndex,
         nextWellSample[wellIndex], nextImage);
 
@@ -976,4 +1095,36 @@ public Channel(String name) {
     }
   }
 
+  class WellIndex implements Comparable<WellIndex> {
+    public int row;
+    public int col;
+
+    public WellIndex(int r, int c) {
+      this.row = r;
+      this.col = c;
+    }
+
+    @Override
+    public int compareTo(WellIndex w) {
+      if (this.row != w.row) {
+        return this.row - w.row;
+      }
+      return this.col - w.col;
+    }
+
+    @Override
+    public boolean equals(Object o) {
+      if (!(o instanceof WellIndex)) {
+        return false;
+      }
+      return compareTo((WellIndex) o) == 0;
+    }
+
+    @Override
+    public int hashCode() {
+      // this would need fixing if we had more than 65535 rows or columns
+      return (row & 0xffff) << 16 | (col & 0xffff);
+    }
+  }
+
 }
diff --git a/src/test/java/com/glencoesoftware/bioformats2raw/test/BioTekTest.java b/src/test/java/com/glencoesoftware/bioformats2raw/test/BioTekTest.java
index cae226ef..2f96b1ba 100644
--- a/src/test/java/com/glencoesoftware/bioformats2raw/test/BioTekTest.java
+++ b/src/test/java/com/glencoesoftware/bioformats2raw/test/BioTekTest.java
@@ -71,7 +71,12 @@ void createPlate(String[] filenames) throws IOException {
     Path testTiff = getTestFile("test.tiff");
 
     for (String f : filenames) {
-      Files.copy(testTiff, input.resolve(f));
+      Path copyLocation = input.resolve(f);
+      if (!input.equals(copyLocation.getParent())) {
+        // supplied file path includes a subdirectory
+        copyLocation.getParent().toFile().mkdirs();
+      }
+      Files.copy(testTiff, copyLocation);
     }
 
     // reset the input path to the first file in the list
@@ -80,16 +85,15 @@ void createPlate(String[] filenames) throws IOException {
   }
 
   /**
-   * Create an artificial single well BioTek plate with the given
-   * list of file names.
+   * Create an artificial BioTek plate with the given list of file names.
    * Checks that the correct well row/column and ZCT sizes are detected.
    *
    * This test will be run once for each Arguments object
    * returned by getTestCases below.
    *
    * @param paths path to each file in the plate
-   * @param wellRow well row index (from 0)
-   * @param wellColumn well column index (from 0)
+   * @param wellRow row index for each well (from 0)
+   * @param wellColumn column index for each well (from 0)
    * @param fields number of fields in the well
    * @param sizeZ number of Z sections
    * @param sizeC number of channels
@@ -97,7 +101,7 @@ void createPlate(String[] filenames) throws IOException {
    */
   @ParameterizedTest
   @MethodSource("getTestCases")
-  public void testBioTek(String[] paths, int wellRow, int wellColumn,
+  public void testBioTek(String[] paths, int[] wellRow, int[] wellColumn,
     int fields, int sizeZ, int sizeC, int sizeT) throws Exception
   {
     // set up the artificial plate
@@ -112,29 +116,33 @@ public void testBioTek(String[] paths, int wellRow, int wellColumn,
       // an exception which would fail the test
       reader.setId(input.toString());
 
-      // the number of OME Images should match the expected field count
+      // the number of OME Images should match the expected well * field count
       // this should be the same as the series count
-      assertEquals(metadata.getImageCount(), fields);
+      assertEquals(metadata.getImageCount(), fields * wellRow.length);
       assertEquals(metadata.getImageCount(), reader.getSeriesCount());
-      // there should be exactly one plate, with exactly one well
+      // there should be exactly one plate
       assertEquals(metadata.getPlateCount(), 1);
-      assertEquals(metadata.getWellCount(0), 1);
+      assertEquals(metadata.getWellCount(0), wellRow.length);
       // the well's row and column indexes should match expectations
       // this is especially important for "sparse" plates where the first
       // row and/or column in the plate are missing
-      assertEquals(metadata.getWellRow(0, 0).getValue(), wellRow);
-      assertEquals(metadata.getWellColumn(0, 0).getValue(), wellColumn);
-      // all of the Images should be linked to the well
-      assertEquals(metadata.getWellSampleCount(0, 0), fields);
-      for (int f=0; f<fields; f++) {
-        // sanity check that the Images are linked to the
-        // well in the correct order
-        assertEquals(metadata.getWellSampleImageRef(0, 0, f), "Image:" + f);
-        // check that the number of Z sections, channels, and timepoints
-        // all match expectations
-        assertEquals(metadata.getPixelsSizeZ(f).getValue(), sizeZ);
-        assertEquals(metadata.getPixelsSizeC(f).getValue(), sizeC);
-        assertEquals(metadata.getPixelsSizeT(f).getValue(), sizeT);
+      for (int w=0; w<wellRow.length; w++) {
+        assertEquals(metadata.getWellRow(0, w).getValue(), wellRow[w]);
+        assertEquals(metadata.getWellColumn(0, w).getValue(), wellColumn[w]);
+        // all of the fields should be linked to the well
+        assertEquals(metadata.getWellSampleCount(0, w), fields);
+        for (int f=0; f<fields; f++) {
+          // sanity check that the Images are linked to the
+          // well in the correct order
+          int imageIndex = (fields * w) + f;
+          assertEquals(metadata.getWellSampleImageRef(0, w, f),
+            "Image:" + imageIndex);
+          // check that the number of Z sections, channels, and timepoints
+          // all match expectations
+          assertEquals(metadata.getPixelsSizeZ(imageIndex).getValue(), sizeZ);
+          assertEquals(metadata.getPixelsSizeC(imageIndex).getValue(), sizeC);
+          assertEquals(metadata.getPixelsSizeT(imageIndex).getValue(), sizeT);
+        }
       }
     }
   }
@@ -175,7 +183,7 @@ static Stream<Arguments> getTestCases() {
     return Stream.of(
       Arguments.of(new String[] {
         "A1_-1_1_1_Tsf[Phase Contrast]_001.tif"
-      }, 0, 0, 1, 1, 1, 1),
+      }, new int[] {0}, new int[] {0}, 1, 1, 1, 1),
       Arguments.of(new String[] {
         "A1_01_1_1_Phase Contrast_001.tif",
         "A1_01_1_2_Phase Contrast_001.tif",
@@ -186,33 +194,111 @@ static Stream<Arguments> getTestCases() {
         "A1_01_1_7_Phase Contrast_001.tif",
         "A1_01_1_8_Phase Contrast_001.tif",
         "A1_01_1_9_Phase Contrast_001.tif",
-      }, 0, 0, 9, 1, 1, 1),
+      }, new int[] {0}, new int[] {0}, 9, 1, 1, 1),
       Arguments.of(new String[] {
         "P24_1_Bright Field_1_001_02.tif"
-      }, 15, 23, 1, 1, 1, 1),
+      }, new int[] {15}, new int[] {23}, 1, 1, 1, 1),
       Arguments.of(new String[] {
         "B2_1_Bright Field_1_001_02.tif"
-      }, 1, 1, 1, 1, 1, 1),
+      }, new int[] {1}, new int[] {1}, 1, 1, 1, 1),
       Arguments.of(new String[] {
         "A1_1_Stitched[AandB_Phase Contrast]_1_001_-1.tif"
-      }, 0, 0, 1, 1, 1, 1),
+      }, new int[] {0}, new int[] {0}, 1, 1, 1, 1),
       Arguments.of(new String[] {
         "A1_1Z0_DAPI_1_001_.tif",
         "A1_1Z1_DAPI_1_001_.tif",
         "A1_1Z2_DAPI_1_001_.tif",
         "A1_1Z3_DAPI_1_001_.tif",
         "A1_1Z4_DAPI_1_001_.tif"
-      }, 0, 0, 1, 5, 1, 1),
+      }, new int[] {0}, new int[] {0}, 1, 5, 1, 1),
       Arguments.of(new String[] {
         "A1_-1_1_1_Stitched[Channel1 300,400]_001.tif",
         "A1_-1_2_1_Stitched[Channel2 500,600]_001.tif",
         "A1_-1_3_1_Stitched[Channel 3 600,650]_001.tif"
-      }, 0, 0, 1, 1, 3, 1),
+      }, new int[] {0}, new int[] {0}, 1, 1, 3, 1),
       Arguments.of(new String[] {
         "A1_-2_1_1_Tsf[Stitched[Channel1 300,400]]_001.tif",
         "A1_-2_2_1_Tsf[Stitched[Channel2 500,600]]_001.tif",
         "A1_-2_3_1_Tsf[Stitched[Channel 3 600,650]]_001.tif"
-      }, 0, 0, 1, 1, 3, 1)
+      }, new int[] {0}, new int[] {0}, 1, 1, 3, 1),
+      Arguments.of(new String[] {
+        "B2/B2_1_Bright Field_1_001_02.tif",
+        "D3/D3_1_Bright Field_1_001_02.tif",
+      }, new int[] {1, 3}, new int[] {1, 2}, 1, 1, 1, 1),
+      Arguments.of(new String[] {
+        "ID_A9 info/A9_-1_1_1_Phase Contrast_001.tif",
+        "ID_A9 info/A9_-1_2_1_GFP_001.tif",
+        "ID_A9 info/A9_-1_3_1_DAPI_001.tif",
+        "ID_A10 info/A10_-1_1_1_Phase Contrast_001.tif",
+        "ID_A10 info/A10_-1_2_1_GFP_001.tif",
+        "ID_A10 info/A10_-1_3_1_DAPI_001.tif",
+        "ID_E9 info/E9_-1_1_1_Phase Contrast_001.tif",
+        "ID_E9 info/E9_-1_2_1_GFP_001.tif",
+        "ID_E9 info/E9_-1_3_1_DAPI_001.tif",
+        "ID_E10 info/E10_-1_1_1_Phase Contrast_001.tif",
+        "ID_E10 info/E10_-1_2_1_GFP_001.tif",
+        "ID_E10 info/E10_-1_3_1_DAPI_001.tif"
+      }, new int[] {0, 0, 4, 4}, new int[] {8, 9, 8, 9}, 1, 1, 3, 1),
+      Arguments.of(new String[] {
+        "A1ROI1_01_1_1Z0_Confocal DAPI_001.tif",
+        "A1ROI1_01_1_1Z1_Confocal DAPI_001.tif",
+        "A1ROI1_01_1_1Z2_Confocal DAPI_001.tif",
+        "A1ROI1_01_1_1Z0_Confocal RFP_001.tif",
+        "A1ROI1_01_1_1Z1_Confocal RFP_001.tif",
+        "A1ROI1_01_1_1Z2_Confocal RFP_001.tif",
+        "A1ROI1_01_1_2Z0_Confocal DAPI_001.tif",
+        "A1ROI1_01_1_2Z1_Confocal DAPI_001.tif",
+        "A1ROI1_01_1_2Z2_Confocal DAPI_001.tif",
+        "A1ROI1_01_1_2Z0_Confocal RFP_001.tif",
+        "A1ROI1_01_1_2Z1_Confocal RFP_001.tif",
+        "A1ROI1_01_1_2Z2_Confocal RFP_001.tif",
+        "A1ROI2_01_1_1Z0_Confocal DAPI_001.tif",
+        "A1ROI2_01_1_1Z1_Confocal DAPI_001.tif",
+        "A1ROI2_01_1_1Z2_Confocal DAPI_001.tif",
+        "A1ROI2_01_1_1Z0_Confocal RFP_001.tif",
+        "A1ROI2_01_1_1Z1_Confocal RFP_001.tif",
+        "A1ROI2_01_1_1Z2_Confocal RFP_001.tif",
+        "A1ROI2_01_1_2Z0_Confocal DAPI_001.tif",
+        "A1ROI2_01_1_2Z1_Confocal DAPI_001.tif",
+        "A1ROI2_01_1_2Z2_Confocal DAPI_001.tif",
+        "A1ROI2_01_1_2Z0_Confocal RFP_001.tif",
+        "A1ROI2_01_1_2Z1_Confocal RFP_001.tif",
+        "A1ROI2_01_1_2Z2_Confocal RFP_001.tif"
+      }, new int[] {0}, new int[] {0}, 4, 3, 2, 1),
+      Arguments.of(new String[] {
+        "A1ROI1_01_1_1Z0_Confocal DAPI_001.tif",
+        "A1ROI1_01_1_1Z1_Confocal DAPI_001.tif",
+        "A1ROI1_01_1_1Z2_Confocal DAPI_001.tif",
+        "A1ROI1_01_1_1Z0_Confocal RFP_001.tif",
+        "A1ROI1_01_1_1Z1_Confocal RFP_001.tif",
+        "A1ROI1_01_1_1Z2_Confocal RFP_001.tif",
+        "A1ROI2_01_1_1Z0_Confocal DAPI_001.tif",
+        "A1ROI2_01_1_1Z1_Confocal DAPI_001.tif",
+        "A1ROI2_01_1_1Z2_Confocal DAPI_001.tif",
+        "A1ROI2_01_1_1Z0_Confocal RFP_001.tif",
+        "A1ROI2_01_1_1Z1_Confocal RFP_001.tif",
+        "A1ROI2_01_1_1Z2_Confocal RFP_001.tif",
+        "A1ROI3_01_1_1Z0_Confocal DAPI_001.tif",
+        "A1ROI3_01_1_1Z1_Confocal DAPI_001.tif",
+        "A1ROI3_01_1_1Z2_Confocal DAPI_001.tif",
+        "A1ROI3_01_1_1Z0_Confocal RFP_001.tif",
+        "A1ROI3_01_1_1Z1_Confocal RFP_001.tif",
+        "A1ROI3_01_1_1Z2_Confocal RFP_001.tif",
+      }, new int[] {0}, new int[] {0}, 3, 3, 2, 1),
+      Arguments.of(new String[] {
+        "A1_01_3_4_Blue_001.tif",
+        "A1_01_3_3_Blue_001.tif",
+        "A1_01_3_2_Blue_001.tif",
+        "A1_01_3_1_Blue_001.tif",
+        "A1_01_2_4_Green_001.tif",
+        "A1_01_2_3_Green_001.tif",
+        "A1_01_2_2_Green_001.tif",
+        "A1_01_2_1_Green_001.tif",
+        "A1_01_1_4_Red_001.tif",
+        "A1_01_1_3_Red_001.tif",
+        "A1_01_1_2_Red_001.tif",
+        "A1_01_1_1_Red_001.tif"
+      }, new int[] {0}, new int[] {0}, 4, 1, 3, 1)
     );
   }